Quantification schemes for quantifying nucleic acids

ABSTRACT

Method and apparatus, including computer program products, implement techniques for quantifying a target nucleic acid (target) in a test sample. The techniques include providing a target and a defined initial amount of an internal control nucleic acid (control) different from the target; amplifying the target and control in a common amplification process; measuring a quantity indicating the amount of amplification product for the target and for the control in relation to a parameter representing the progress of the amplification; determining a characteristic value of the progress parameter for the target based on measurement results related to the amount of target amplification product; possibly determining a characteristic value of the progress parameter for the control based on measurement results related to the amount of control amplification product; and quantifying the original amount of target according to a predefined or selected quantification scheme using at least the characteristic value for the target.

TECHNICAL FIELD

This invention relates to the quantification (quantitation) of nucleicacids.

BACKGROUND

Methods for the quantification (quantitation) of nucleic acids areimportant in many areas of molecular biology and in particular formolecular diagnostics. At the DNA level such methods are used forexample to determine the copy numbers of gene sequences amplified in thegenome. Further, methods for the quantification of nucleic acids areused in connection with the determination of mRNA quantities since thisis usually a measure for the expression of the respective coding gene.

Among the number of different analytical methods that detect andquantify nucleic acids or nucleic acid sequences, Polymerase ChainReaction (PCR) has become the most powerful and widespread technology,the principles of which are disclosed in U.S. Pat. Nos. 4,683,195 and4,683,202 (Mullis et al.). However, a typical PCR reaction by itself andcorrespondingly a typical reverse-transcriptase PCR (RT-PCR) reaction byitself only yields qualitative data, since, after a phase of exponentialor progressive amplification, the amount of amplified nucleic acidreaches a plateau, such that the amount of generated reaction product isnot proportional to the initial concentration of the target nucleic acidor target nucleic acid sequence, in particular template DNA. However, anend point analysis reveals the presence or absence of a respectivestarting nucleic acid. This information is, for certain applications, inparticular clinical applications, of high value. For other applicationsincluding clinical applications, a quantitative measurement is needed,for example to make a proper diagnosis with respect to certain diseases.For example, precise quantitative measurements are needed to diagnosecertain infectious diseases, cancers and autoimmune diseases. It may beuseful therapeutically to assess the response of a disease to treatmentand make prognoses for recovery. Precise quantitative measurement mayalso help detect false positives, which can occur if there is anycontamination of a sample.

SUMMARY

The invention provides a method for quantification of at least onetarget nucleic acid in a test sample or in a plurality of test samples.In general, in one aspect, the method comprises: providing at least onetarget nucleic acid together with at least one internal control in acommon test sample, the internal control comprising a defined initialamount of a control nucleic acid different from the target nucleic acid;amplifying the target nucleic acid and the control nucleic acid withinthe test sample in a common nucleic add amplification process; directlyor indirectly measuring the amount of amplification product or aquantity indicating the amount of amplification product for the targetnucleic add and the control nucleic acid during the amplification inrelation to an increasing progress parameter representing the progressof the amplification process; determining a characteristic value of theprogress parameter for the target nucleic acid on the basis ofmeasurement results related to the amount of amplification product forthe target nucleic acid; at least for certain cases: determining acharacteristic value of the progress parameter also for the controlnucleic acid on the basis of measurement results related to the amountof amplification product for the control nucleic acid; quantifying theoriginal amount of target nucleic acid in the test sample according topredefined or selected quantification scheme on the basis of at leastthe characteristic value determined for the target nucleic acid.

In particular implementations, the method further comprises the step ofselecting between a plurality of quantification schemes of differenttypes, wherein at least one quantification scheme provides forqualification of the original amount of target nucleic acid in the testsample without reference to any characteristic value for the controlnucleic acid and at least one quantification scheme provides forquantification of the original amount of target nucleic acid in the testsample with reference to the characteristic value for the controlnucleic acid.

The details of one or more embodiments of the invention are set forth inthe accompanying drawings and the description below. Other features,objects, and advantages of the invention will be apparent from thedescription and drawings, and from the claims.

DESCRIPTION OF DRAWINGS

FIGS. 1A and B show a method for calibrating the quantification ofnucleic acids on the basis of PCR amplification using a calibrationformula based on a standard.

FIG. 2 shows an exemplary PCR amplification growth curve before (FIG. 2a) and after (FIG. 2 b) normalization of the fluorescence measurementresults.

FIGS. 3 to 8 show diagrams and plots including numerical examples for acalibration effected in agreement with FIGS. 1 a, 1 b.

FIG. 9 shows two possibilities for a calibration on the basis of astandard for high target concentrations.

FIGS. 10 a and 10 b show an example for a standard quantification on thebasis of amplification results referring to the target and an internalcontrol.

FIGS. 11 a and 11 b show two possibilities for an exceptionalquantification on the basis of amplification results referring to thetarget only.

FIG. 12 shows the print on the packet of a HBV test kit used to evaluatethe invention and to provide illustrative experimental results shownherein.

FIG. 13 shows growth curves for a dilution series of HBV plasmid targetand internal controls obtained on basis of the HBV test according toFIG. 12.

FIG. 14 indicates respective elbow values for the target growth curvesof the example in FIG. 13.

FIG. 15 shows growth curves for a second dilution series (secondresearch lot) of HBV plasmid target and internal controls obtained onbasis of the HBV test according to FIG. 12.

FIG. 16 indicates respective elbow values for the target growth curvesof the example in FIG. 15.

FIGS. 17 a and 17 b give examples for calibration formulas, which canalternatively be used for the quantification of the initial targettiter.

FIG. 18 is a flow chart showing one possibility how the selectionbetween alternate quantification formulas can be effected.

FIG. 19 is a flow chart showing another possibility how the selectionbetween alternate quantification formulas can be effected.

FIG. 20 shows the titer calculation results for the growth curve of FIG.13.

FIG. 21 shows the signal recovery results for the growth curve of FIG.13.

FIG. 22 shows the titer calculation results for the growth curve of FIG.15.

FIG. 23 shows the signal recovery results for the growth curve of FIG.15.

FIG. 24 shows schematically the basic structure of a quantificationapparatus, which might be used for performing the method according tothe invention.

Like reference symbols in the various drawings indicate like elements.

DETAILED DESCRIPTION

In order to obtain reliable and reproducible quantitative data, manydifferent PCR based protocols have been developed. Generally, twodifferent basic principles can be discriminated conventionally, namelyi) competitive PCR (or RT-PCR) using internal standards and ii)quantification of target DNA by initial generation of a calibrationcurve. It is referred in this respect to Siebert, Molecular Diagnosis ofInfectious Diseases (ed. Udo Reischl, Humana Press, Totowa, N.J., p.55-79, 1998).

The competitive PCR or RT-PCR allows an end point determination of theamount of PCR product formed in the plateau phase of the amplificationreaction. For example, a specific target sequence is coamplifiedtogether with a dilution series of an internal standard of a known copynumber. The initial copy number of the target sequence is extrapolatedfrom the mixture containing an identical PCR product quantity ofstandard and target sequence (Zimmermann and Mannhalter, BioTechniques21:280-279, 1996). A disadvantage of this method is that the measurementoccurs in the saturation region of the amplification reaction.

A major improvement in the generation of quantitative data derives fromthe possibility of measuring the kinetics of a PCR reaction by on-linedetection. This has become possible by means of detecting the ampliconthrough fluorescence monitoring. Examples of such techniques aredisclosed in detail in WO 97/46707, WO 97/46712 and WO 97/46714 (Wittweret al.).

Higuchi et al. (BioTechnology 11, 1026-2030, 1993) disclosed an approachfor initial template quantification using fluorescence monitoring ateach cycle. A fluorescence threshold level was used to define afractional cycle number related to initial template concentration.Specifically, the log of the initial template concentration was found tobe inversely proportional to the fractional cycle number (CT), definedas the intersection of the fluorescence versus cycle number curve withthe fluorescence threshold.

Kinetic real-time quantification in the exponential phase of PCR can beeffected on basis of an internal standard as well as of an externalstandard. Generally, the formation of PCR products is monitored in eachcycle of the PCR. The amplification is usually measured in thermocyclersthat have additional devices for measuring fluorescence signals duringthe amplification reaction. A typical example of this is the RocheDiagnostics Light Cycler (Cat. No. 2 0110468).

The amplification products are for example detected by means offluorescent-labeled hybridization probes that only emit fluorescencesignals when they are bound to the target nucleic acid or in certaincases also by means of fluorescent dyes that bind to double-strandedDNA. A defined signal threshold is determined for all reactions to beanalyzed and the number of cycles, cp, required to reach this thresholdvalue is determined for the target nucleic acid and—if an internalstandard or internal control (possibly housekeeping gene) is used—forthe reference nucleic acid. The absolute or relative copy numbers of thetarget molecule can be determined on basis of the cp values obtained forthe target nucleic acid and the reference nucleic acid or—where anexternal standard is used—on basis of the respective cp value obtainedfor the target molecule and a standard curve constructed on basis ofseparate amplification results for the reference nucleic acid (e.g.dilution series of the reference nucleic acid) (Gibson et al., GenomeResearch 6:995-1001, 1996; Bièche et al., Cancer Research 59:2759-2765,1999; WO 97/46707; WO 97/46712; WO 97/46714).

The use of external standards has the advantage that the standard andtarget nucleic acid are amplified in separate reaction vessels. In thiscase a standard can be used which has an identical sequence to thetarget nucleic acid. However, systematic errors can occur in this typeof quantification, for example due to inhibitory components that impairthe efficiency of the subsequent PCR reaction. These errors can beexcluded by using internal standards, i.e. by amplifying the standardand target nucleic acid in one reaction vessel. However, a disadvantageof this method is that standards have to be used that have differentsequences compared to the target nucleic acid to be analyzed in order tobe able to distinguish between the amplification of the standard andtarget nucleic acid. This can lead to systematic errors in thequantification as well, since different efficiencies of the PCRapplication cannot be excluded when the sequences are different.

With respect to the issue use of internal or external controls orstandards we distinguish the following approaches:

a) External controls (the term external standards is also appropriate)are amplified by subjecting different amounts of a known DNA to PCR toobtain a standard curve. The amount of sample DNA amplified separately,possibly in parallel, can be derived directly by interpolation of thestandard curve. Preconditions for this quantification method are thatthe efficiency of both PCRs, that of the standard DNA and that of thesample DNA, are sufficiently identical and that the amplificationreactions are in the exponential phase.

b) PCRs using internal controls (IC) or internal standards (IQS,Internal Quantification Standards) can be performed as multiplex orcompetitive PCR. In multiplex PCR the primer pair for the IC is derivedfrom a different DNA molecule than the primer pair for sample DNAwhereas in competitive PCR the same primer pair is used for both, the ICand sample DNA. Preconditions for quantification by multiplex PCR arethe same as for PCRs using external controls. Multiplex PCR as comparedto PCRs using external standards has, however, the advantage, that bothIC DNA and sample DNA are amplified in the same tube and thus problemsdue to variations in tube consistency or position in the thermal cyclerare eliminated. In competitive PCR, amplicons generated from the IC andsample DNA by the same primer pair have to be differentiated. During theamplification both IC DNA and sample DNA compete for the same primerpair. If the amount of initial template DNAs (IC and sample DNA) isunequal, PCR product yield is shifted to the credit of the DNA being inexcess. Only when the amount of initial template DNA is equimolar,product yield for IC and sample DNA is identical, as far as identicalamplification efficiency applies.

As already indicated, the possibility of measuring the kinetics of anamplification reaction has become enormously facilitated since there areinstruments and methods available, wherein the generation of theamplification product can be measured continuously by spectroscopicdetection principles, in particular by means of fluorescence. An examplefor a suitable instrument is described in detail in Wittwer et al.,BioTechniques 22, No. 1, 176-181 (1997).

Several detection formats that are based on target dependent fluorescentsignaling, and which enable continuous monitoring of the generation ofamplification products, have been disclosed (reviewed in Wittwer et al.,BioTechniques, 22, No. 1, 130-138, 1997). These detection formatsinclude but are not limited to:

1. Use of Fluorescent Double-stranded DNA Recognizing Compounds

-   -   Since the amount of double-stranded amplification product        usually exceeds the amount of nucleic acid originally present in        the sample to be analyzed, double-stranded DNA specific dyes can        be used, which upon excitation with an appropriate wavelength        show enhanced fluorescence only if they are bound to        double-stranded DNA. Dyes that do not affect the efficiency of        the PCR reaction, for example, SYBR Green I, are used        advantageously.

2. Increased Fluorescence Resonance Energy Transfer upon Hybridization

-   -   For this detection format, two oligonucleotide hybridization        probes each labelled with a fluorescent moiety are used which        are capable of hybridizing to adjacent but not overlapping        regions of one strand of the amplification product. When        hybridized to the target DNA, the two fluorescent labels are        brought into close contact, such that fluorescence resonance        energy transfer (FRET) between the two fluorescent moieties can        take place. As a consequence, the hybridization can be monitored        through excitation of the donor moiety and subsequent        measurement of fluorescence emission of the second acceptor        moiety.    -   In a similar embodiment, only one fluorescently labelled probe        is used, which together with one appropriately labelled primer        may also serve as a specific FRET pair (Bernard et al.,        Analytical Biochemistry 255, p. 101-107, 1998).

3. Detection Principle as Used in the TaqMan™ Instrument

-   -   In order to detect the amplification product, a single-stranded        hybridization probe is used, which is labelled with a        fluorescent entity, the fluorescence emission of which is        quenched by a second label on the same probe which may act as a        quenching compound on basis of Förster-type energy transfer        effects. During the annealing step of the PCR reaction, the        probe hybridizes to its target sequence, and, subsequently,        during the extension of the primer, the DNA polymerase having a        5′-3′-exonuclease activity digests the hybridization probe into        smaller pieces, such that the fluorescent entity is separated        from the quencher compound. After appropriate excitation,        fluorescence emission can be monitored as an indicator of        accumulating amplification product.

4. Molecular Beacons

-   -   Similar to the probes/formats used in the TaqMan™ instrument, a        molecular beacon oligonudeotide is labelled with a fluorescent        compound and a quencher compound, which due to the secondary        structure of the molecule are in close vicinity to each other.        Upon binding to the target DNA, the intramolecular hydrogen        bonding is broken, and the fluorescent compound located at one        end of the probe is separated from the quencher compound, which        is located at the opposite end of the probe (Lizardi et al.,        U.S. Pat. No. 5,118,801).

A method for determining an unknown starting quantity of a targetnucleic acid sequence in a test sample is known from US 2002/0031768 A1(McMillan et al.). The unknown starting quantity of the target nucleicacid sequence in the test sample and known starting quantities of acalibration nucleic acid sequence in respective calibration samples areamplified. For each of the nucleic acid sequences a respective thresholdvalue is determined, using a derivative of a growth curve derived forthe sequence. Using the threshold value determined for the targetsequence and a calibration curve derived from the threshold valuesdetermined for the calibration nucleic acid sequence the startingquantity of the target nucleic acid is determined. McMillan et al.discloses also instrumentation and apparatus that can be used for theimplementation of the method. Further, methods for determining astarting quantity of a nucleic acid sequence in a sample usingquantitative internal controls or using internal standards aredisclosed, as for example in US 2002/0034745 A1, US 2002/0034746 A1 andUS 2002/0058282 A1.

The quantification of analytes on basis of a derivative calculated froma growth curve is also known from EP 1 041 158 A2 (compare also U.S.Pat. Nos. 6,303,305 B1, 6,503,720 B2 and US 2002/0028452 A1, Wittwer etal.).

EP 1 138 783 A2 and EP 1 138 784 A2 (compare also US 2002/0058262 A1,Sagner et al.) disclose methods for absolute and relative quantificationof a target nucleic acid involving the determination of an amplificationefficiency. It is referred to internal standards as well as to externalstandards.

U.S. Pat. No. 5,389,512 (Sninsky et al.) discloses a method fordetermining the relative amount of a nucleic acid sequence in a sampleby the polymerase chain reaction. The method involves the simultaneousamplification of the nucleic acid segment and a second nucleic acidsegment present in a sample. The amount of amplified DNA from eachsegment is determined and compared to standard curves to determine theamount of the nucleic acid segment present in the sample before theamplification expressed as a ratio of the first segment to secondsegment. Two standard curves are used, one referring to theamplification of the first segment and the other referring to theamplification of the second segment.

U.S. Pat. No. 5,476,774 (Wang et al.) provides a further method fordetermining the amount of a target acid segment in a sample bypolymerase chain reaction. The method involves the simultaneousamplification of the target nucleic acid segment and an internalstandard nucleic acid segment. The amount of amplified DNA from eachsegment is determined and compared to standard curves to determine theamount of the target nucleic acid segment present in the sample prior toamplification.

Instrumentation and apparatus which can be used advantageously in thecontext of PCR-quantification are disclosed in various publications,e.g. EP 0 953 379 A1, disclosing an apparatus for simultaneouslymonitoring reactions taking place in a plurality of reaction vessels, EP0 953 837 A1, disclosing a fluorescence light measuring device which canbe used in such an apparatus, and EP 0 955 097 A1, disclosing a thermalcycler for performing amplification of nucleic acids.

The above-mentioned documents are completely incorporated by referencein the disclosure of the present application. Further, it is referred inthis respect to Ullmann's Encyclopedia of Industrial Chemistry, 6. ed.,Vol. 28, section “Polymerase Chain Reaction” (pages 125 to 159), inparticular chapter 10 “Quantitative PCR” (pages 140 to 144,WILEY-VCH-Verlag, Weinheim, Germany 2003), which is also incorporated byreference in its entirety.

The invention refers to, and, according to a first, more general,aspect, provides a method for quantification of at least one targetnucleic acid in a test sample or in a plurality of test samples whichcomprises the steps of:

-   -   providing at least one target nucleic acid together with at        least one internal control in a common test sample, said        internal control comprising a defined initial amount of a        control nucleic acid different from said target nucleic acid;    -   amplifying said target nucleic acid and said control nucleic        acid within said test sample in a common nucleic acid        amplification process;    -   measuring the amount of amplification product or a quantity        indicating the amount of amplification product for said target        nucleic acid and said control nucleic acid during said        amplification in relation to an increasing progress parameter        representing the progress of said amplification process;    -   determining a characteristic value of said progress parameter        for said target nucleic acid on basis of measurement results        related to the amount of amplification product for said target        nucleic acid;    -   determining a characteristic value of said progress parameter        also for said control nucleic acid on basis of measurement        results related to the amount of amplification product for said        control nucleic acid;    -   quantifying the original amount of target nucleic acid in said        test sample according to a predetermined quantification scheme,        said quantification scheme providing for the quantification of        the original amount of target nucleic acid in said test sample        on basis of the characteristic value for said target nucleic        acid, the characteristic value for said control nucleic acid and        associated reference data.

Measuring the amount of amplification product or a quantity indicatingthe amount of amplification product can be done directly, for example,by determining a characteristic of the amplification product, orindirectly, for example, by measuring a quantity that is associated toor correlated with the amount of amplification product.

This method for quantification allows the use of an external standard incombination with an internal control or standard, so that on the onehand the standard nucleic acid can have the same sequence as the targetnucleic acid and on the other hand changes between the efficiencies ofthe respective PCR reaction in different reaction vessels can be takeninto account to a certain extent on basis of the internal control orstandard. Advantageous embodiments of the above defined method resultimplicitly from the following explanations.

It was observed that the amplification of different nucleic acids withina common sample in a common nucleic acid amplification process can leadto effects and problems which are caused by the competition between theindividual amplification reactions with respect to the different nucleicacids. With respect to PCR and RT-PCR there will be competition fornudeotides, enzymes and other ingredients contained in the PCR reactionmix and, in the competitive PCR, when the respective nucleic acids haveidentical primer binding sides, for the primers. In particular,competitive amplification of an internal quantification standard orinternal control and the target within a method for quantification canlead to decreased sensitivity of the method or even to failure of thequantification of the original amount of target according to aquantification scheme used when there is a strong imbalance between theamounts of target and internal control at the beginning of theamplification reaction, in particular PCR reaction. Either the internalcontrol of the target will be detected with decreased sensitivity orwill not be detected in an extent that allows quantification. Forexample, there can be a dropout of target growth curves, if theconcentration of the internal control concentration is too high, orthere can be a dropout of internal control growth curves, if the targetconcentration is too high. Accordingly, the dynamic range of the methodand a corresponding system is limited. Targeted specifications for thelinear range can only be met either for a lower range or an upper rangeon basis of certain reagent lots. Of course, it is possible to providedifferent reagent lots aiming to the lower range and to the upper range.This solution, however, is cumbersome from a practical point of view,and there remains a considerable risk to fail at least at one end (thelower or the upper) due to the labile situation at thequantification/detection limits.

According to a first, more general, aspect, it is an object of theinvention to provide a method for quantification of at least one targetnucleic acid in a test sample or a plurality of test samples which isless prone to systematic errors than methods of the prior art. Thisobject is achieved by the method defined in the foregoing.

According to a second, more specific, aspect, it is an object of theinvention to provide a method for quantification of at least one targetnucleic acid in a test sample or in a plurality of test samples that hasan extended dynamic range.

To solve both objects, in one implementation, a method forquantification of at least one target nucleic acid in a test sample orin a plurality of test samples comprises the steps of:

a) providing at least one target nucleic acid together with at least oneinternal control in a common test sample, said internal controlcomprising a defined initial amount of a control nucleic acid differentfrom said target nucleic acid;

b) amplifying said target nucleic acid and said control nucleic acidwithin said test sample in a common nucleic acid amplification process;

c) directly or indirectly measuring the amount of amplification productor a quantity indicating the amount of amplification product for saidtarget nucleic acid and said control nucleic acid during saidamplification in relation to an increasing progress parameterrepresenting the progress of said amplification process;

d) determining a characteristic value of said progress parameter forsaid target nucleic acid on basis of measurement results related to theamount of amplification product for said target nucleic acid;

e) if certain predefined conditions referring to the measured amount ofamplification product for said control nucleic acid apply, determining acharacteristic value of said progress parameter also for said controlnucleic acid on basis of measurement results related to the amount ofamplification product for said control nucleic acid;

f) selecting between a plurality of quantification schemes according toat least one predefined selection criterion, said selection beingeffected directly or indirectly on basis of at least one of saidmeasurement results related to the amount of amplification product forsaid target nucleic acid, measurement results related to the amount ofamplification product for said control nucleic acid and saidcharacteristic value or values,

wherein at least one quantification scheme of a first type provides fora quantification of the original amount of target nucleic acid in saidtest sample on basis of the characteristic value for said target nucleicacid, the characteristic value for said control nucleic acid andassociated reference data,

wherein at least one quantification scheme of a second type provides fora quantification of the original amount of target nucleic acid in saidtest sample on basis of the characteristic value for said target nucleicacid and associated reference data without reference to anycharacteristic value for said control nucleic acid,

g) quantifying the original amount of target nucleic acid in said testsample according to the selected quantification scheme on basis of atleast the characteristic value determined for said target nucleic acid.

In this implementation, the dynamic range can be extended by usingdifferent quantification schemes for different ranges, for example aquantification scheme of the first type for a lower and medium rangeand, if necessary or appropriate, a quantification scheme of the secondtype for an upper range. In particular, a dropout of the internalcontrol growth curve in situations where the target concentration is toohigh does not affect a quantification scheme of the second type.

Dependent on the details of the quantification scheme of the second typeand the associated reference data used, it is in principle possible toobtain a quantification of a quality that corresponds more or less tothe quality of quantification conventionally obtained on basis of aquantification using an external standard and no internal control. Ifthe quantification scheme or quantification schemes of the second typerefer to an upper range of the targeted linear range of thequantification method, then the situation on a whole is even better,since—in the context of PCR amplification—high target concentrationsmean fewer PCR cycles to achieve a certain target yield, so that changesin the amplification factor per PCR cycle are less relevant. To say itdifferently: Since the accuracy of a titer measurement scales inverselywith the number of PCR cycles, the error of high target yields obtainedon basis of high initial target concentrations is relatively small,because fewer cycles are involved in the quantification process. Ofcourse, the advantages of the use of an internal control such as anadditional validation by considering the internal control amplificationresults will not be obtained on basis of the quantification scheme ofthe second type. This, however, is in any case better than obtaining noquantification or a completely wrong quantification.

In another implementation, a quantification scheme of the second type isselected when at least one of the following conditions apply:

i) no characteristic value for said control nucleic acid was determined,

ii) the characteristic value for said control nucleic acid exceeds apredefined threshold value,

iii) the characteristic value for said target nucleic acid falls shortof a predefined threshold value,

iv) the characteristic value for said control nucleic acid exceeds thecharacteristic value for said target nucleic acid by at least apredefined amount,

v) the amount of amplification product or the quantity indicating theamount of amplification product for said control nucleic acid asmeasured or estimated for a final stage or near final stage of theamplification process falls short of a predefined minimum plateau value,

vi) the amount of amplification product or the quantity indicating theamount of amplification product for said control nucleic acid asmeasured or estimated for a momentary state of the amplification processassociated to the characteristic value for the target nucleic acid fallsshort of a predefined threshold value, the amount of amplificationproduct, or the quantity indicating the amount of amplification productfor said target nucleic acid as measured or estimated for said momentarystate of the amplification process.

According to one approach, a quantification scheme of the first type isselected when a characteristic value for said control nucleic acid wasdetermined. Alternatively, a quantification scheme of the first type canbe selected when a characteristic value for said control nucleic acidwas determined and at least one of the following conditions apply:

ii) the characteristic value for said control nucleic acid falls shortof a predefined threshold value,

iii) the characteristic value for said target nucleic acid exceeds apredefined threshold value,

iv) the characteristic value for said control nucleic acid falls shortof a threshold value defined in relation and greater than thecharacteristic value for said target nucleic acid,

v) the amount of amplification product or the quantity indicating theamount of amplification product for said control nucleic acid asmeasured or estimated for a final stage or near final stage of theamplification process exceeds of a predefined minimum plateau value,

vi) the amount of amplification product or the quantity indicating theamount of amplification product for said control nucleic acid asmeasured or estimated for a momentary state of the amplification processassociated to the characteristic value for the target nucleic acidexceeds a predefined threshold value, the amount of amplificationproduct, or the quantity indicating the amount of amplification productfor said target nucleic acid as measured or estimated for said momentarystate of the amplification process.

The reference data associated to said or respective quantificationscheme of the first type can be calibration data determined or providedby

A) providing at least one standard together with at least one internalcontrol in a common sample, said standard comprising a defined initialamount of a standard nucleic acid, said internal control comprising adefined initial amount of a control nucleic acid, said standard nucleicacid and said control nucleic acid being different;

B) amplifying said standard and said internal control within said samplein a common nucleic acid amplification process;

C) directly or indirectly measuring the amount of amplification productor a quantity indicating the amount of amplification product for saidstandard nucleic acid and said control nucleic acid during saidamplification in relation to an increasing progress parameterrepresenting the progress of said amplification process;

D) determining a characteristic value of said progress parameter forsaid standard nucleic acid on basis of measurement results related tothe amount of amplification product for said standard nucleic acid;

E) determining a characteristic value of said progress parameter forsaid control nucleic acid on basis of measurement results related to theamount of amplification product for said control nucleic add;

H) relating said initial amount of standard nucleic acid on the one handand said characteristic values on the other hand with reference to saidquantification scheme of the first type to provide said calibration dataassociated to said quantification scheme of the first type.

With respect to the reference data associated to said or respectivequantification scheme of the second type two approaches arecontemplated. According to a first approach the reference data arecalibration data determined or provided on basis of

AA) providing at least one standard in a sample, said standardcomprising a defined initial amount of a standard nucleic acid;

BB) amplifying said standard in a nucleic acid amplification process;

CC) directly or indirectly measuring the amount of amplification productor a quantity indicating the amount of amplification product for saidstandard nucleic acid during said amplification in relation to anincreasing progress parameter representing the progress of saidamplification process;

DD) determining a characteristic value of said progress parameter forsaid standard nucleic acid on basis of measurement results related tothe amount of amplification product for said standard nucleic acid;

HH) relating said initial amount of standard nucleic acid on the onehand and said characteristic value on the other hand with reference tosaid quantification scheme of the second type to provide saidcalibration data associated to said quantification scheme of the secondtype.

According to a second approach, said reference data associated to saidor respective quantification scheme of the second type are calibrationdata determined or provided on basis of steps A) to D) and on basis of

HH) relating said initial amount of standard nucleic acid on the onehand and said characteristic value associated to said standard nucleicacid on the other hand with reference to said quantification scheme ofthe second type to provide said calibration data associated to saiddefined initial amount of said control nucleic acid being amplifiedtogether with said standard nucleic acid can correspond to said definedinitial amount of said control nucleic add being amplified together withsaid target nucleic add. Said control nucleic add, which is amplifiedtogether with said standard nucleic acid, can correspond to said controlnucleic add which is amplified together with said target nucleic acid.Said standard can be an external standard, said sample being differentfrom said test sample. Said standard nucleic acid can correspond to saidtarget nucleic acid.

At least one and possibly all of said steps A) to H) and possibly HH)or/and at least one or possibly all of said steps AA) to HH) can beeffected simultaneously to respective steps a) to g).

Steps A) to E) or/and steps AA) to DD) can be effected before or aftereffecting steps a) to e). Steps A) to H) and possibly HH) or/and stepsAA) to HH) can be effected before effecting steps a) to g). For example,said steps can be effected by the manufacturer of a quantification kit.In particular, said calibration data associated to said quantificationscheme of the first type or/and said calibration data associated to saidquantification scheme of the second type can be provided together withconstituents of a quantification kit.

In step A) a dilution series of said standard nucleic acid can beprovided, each dilution within a respective sample together with saidinternal control, that steps B) to E) are effected with respect to allsamples of said dilution series, and that step H) comprises:

-   -   relating the initial amounts of standard nucleic acid of said        samples on the one hand and the characteristic values determined        for said samples on the other hand with reference to said        quantification scheme of the first type to provide said        calibration data associated to said quantification scheme of the        first type.

Further, step HH), can comprise:

-   -   relating the initial amount of standard nucleic acid of a        selected or predefined one of said samples on the one hand and        the characteristic value associated to said standard nucleic        acid determined for said sample being selected on the other hand        with reference to said quantification scheme of the second type        to provide said calibration data associated to said        quantification scheme of the second type.

Alternatively step HH) can comprise:

-   -   relating the initial amounts of standard nucleic acid of said        samples on the one hand and the characteristic values associated        to said standard nucleic acid determined for said samples on the        other hand with reference to said quantification scheme of the        second type to provide said calibration data associated to said        quantification scheme of the second type.

In step AA) only one sample including a selected defined initial amountof said standard nucleic acid is provided. However, it can beadvantageous, if in step AA) a dilution series of said standard nucleicacid is provided, each dilution within a respective sample, whereinsteps BB) to DD) are effected with respect to all samples of saiddilution series, and wherein step HH) comprises:

-   -   relating the initial amounts of standard nucleic of said samples        on the one hand and the characteristic values determined for        said samples on the other hand with reference to said        quantification scheme of the second type to provide said        calibration data associated to said quantification scheme of the        second type.

Irrespective of how the calibration data associated to saidquantification scheme of the second type are provided, said calibrationdata can include a fixed amplification efficiency. Alternatively, instep g) the calibration data associated to said quantification scheme ofthe second type can be used together with a fixed amplificationefficiency for the quantification of the original amount of targetnucleic in said test sample according to the quantification scheme ofthe second type.

Generally, a theoretical amplification efficiency of said amplificationprocess can be used as said fixed amplification efficiency. Said fixedamplification efficiency can be determined on basis of steps A) to H) oron basis of steps AA) to HH), said fixed amplification efficiency beingincluded in or derived from said calibration data associated to saidquantification scheme of the first type or being included in saidcalibration data associated to said quantification scheme of the secondtype.

The determination of the fixed amplification efficiency on basis ofamplification results can lead to more precise quantification on basisof the quantification scheme of the second type.

Generally, said amplification process (or the respective amplificationprocess) can be effected in cycles. This is in particular true forconventional PCR and RT-PCR processes already referred to. If said (therespective) amplification process is effected in cycles, a cycle numberindicating the number of elapsed cycles can be used as progressparameter. However, irrespectively whether said amplification process iseffected in cycles or not, it is possible to use an elapsed time ofamplification as progress parameter. With reference to step c) and alsothe corresponding step of the solution according to the first, moregeneral, aspect this step can comprise:

-   -   measuring, at a plurality of different times during        amplification, at least one signal whose intensity is related to        the quantity of the respective nucleic acid being amplified.

This is also suggested with respect to step C) and with respect to stepCC). The signal being measured can be an optical signal, for examplefluorescence radiation emitted from fluorescent entities, in particulardyes, associated to the respective nucleic acid, fluorescent labelhybridization probes associated to the respective nucleic acid or thelike.

Further, at least one of step d) and step e) and of the correspondingsteps of the solution according to the first, more general, aspect cancomprise or be based on:

-   -   deriving a growth curve from the respective measurement results,        possibly the measurements of the respective signal.

This is also suggested with respect to at least one of step D) and E)and with respect to step DD).

Further, at least one of step d) and step e) and at least one of thecorresponding steps of the solution according to the first, moregeneral, aspect can comprise:

-   -   identifying a characteristic of the respective growth curve or        of a derivative calculated of the respective growth curve,    -   determining the characteristic value associated with the        respective characteristic.

This is also suggested with respect to at least one of step D) and stepE) and with respect to step DD).

The characteristic of the respective growth curve can correspond to acrossing of a threshold by the growth curve, said threshold beingpredefined to represent an unnormalized growth value or being determinedon basis of respective measurement results to represent a normalizedgrowth value. If such a characteristic of the growth curve (for example,a normalized growth curve) is used, then it is generally not necessaryto calculate a derivative of the growth curve.

With respect to the possibility to use a derivative calculated of therespective growth curve, it is suggested that the characteristic of therespective derivative corresponds to a positive peak or a negative peakor a zero crossing of the derivative. The derivative can be the first orsecond derivative of the respective growth curve.

The determination of the respective characteristic value can involve theinterpolation of the growth curve between growth curve pointsrepresenting a respective measurement, or the interpolation of acalculated derivative curve between respective points, to give acharacteristic value not necessarily corresponding to a value of theprogress parameter for which a measurement was effected. In particular,a fractional cycle number can be determined as characteristic value.

Generally, said characteristic value or values represent a direct orindirect measure of at least one of the amplification and the originalamount or defined initial amount of the respective nucleic acid.

In one implementation, according to said or at least one quantificationscheme of the first type, a secondary characteristic value is determinedfrom said characteristic value for said target nucleic acid or standardnucleic acid, respectively, and said characteristic value of saidcontrol nucleic acid. The secondary characteristic value represents adirect or indirect measure of at least one of the amplification and theoriginal amount of said target nucleic acid or initial amount of saidstandard nucleic acid, respectively, relative to at least one of theamplification and the defined initial amount of said control nucleicacid. In this case the original amount of target nucleic acid can bequantified on basis of said secondary characteristic value and saidreference data associated thereto.

It is further suggested that step H) comprises:

-   -   relating said initial amount of standard nucleic acid on the one        hand and said secondary characteristic value on the other hand        with reference to said quantification scheme of the first type        to provide said calibration data associated to said        quantification scheme of the first type.

According to said or at least one quantification scheme of the firsttype, a ratio value representing the ratio of the characteristic valuefor said target nucleic acid or standard nucleic acid, respectively, andthe characteristic value of said control nucleic acid can be determined.In this case the original amount of target nucleic acid can bequantified on basis of said ratio value and said reference dataassociated thereto.

In this context, step H) can comprise:

-   -   relating said initial amount of standard nucleic acid on the one        hand and said ratio value on the other hand with reference to        said quantification scheme of the first type to provide said        calibration data associated to said quantification scheme of the        first type.

According to said or at least one quantification scheme of the firsttype, a difference value representing the difference between thecharacteristic value for said target nucleic acid or standard nucleicacid, respectively, and the characteristic value of said control nucleicacid can be determined. In this case, the original amount of targetnucleic acid can be quantified on basis of said difference value andsaid reference data associated thereto.

In this context it is further suggested said step H) comprises:

-   -   relating said initial amount of standard nucleic acid on the one        hand and said difference value on the other hand with reference        to said quantification scheme of the first type to provide said        calibration data associated to said quantification scheme of the        first type.

A plurality of secondary characteristic values (e.g. ratio values ordifference values) can be used, which correspond to the secondarycharacteristic values of the samples of the dilution series. In thiscase step H) may comprise:

-   -   relating said initial amounts of standard nucleic acid of said        samples on the one hand and said secondary characteristic values        determined for said samples on the other hand with reference to        said quantification scheme of the first type to provide said        calibration data associated to said quantification scheme of the        first type.

To provide said calibration data generally a calibration equation orcalibration formula can be used which represents a relation between theinitial amount of said standard nucleic acid, the initial amount of saidinternal control and said characteristic values (i.e. the characteristicvalue of said progress parameter for said standard nucleic acid and thecharacteristic value of said progress parameter for said control nucleicacid) or the secondary characteristic value.

With reference to PCR amplification the relation between initial numbersof nucleic acid molecules and the amplified product is described by anexponential function:y=y ₀(1+ε)^(n)

with

-   -   y=product yield (amount of DNA after amplification)    -   y₀=amount of template DNA prior to PCR    -   ε=efficiency of the amplification process    -   n=number of cycles

Denoting the product yield for the target or standard nucleic acid as T,the initial amount of target or standard nucleic acid prior to PCR asT₀, the efficiency of the PCR amplification for the target or standardnucleic acid as ε_(T), the cycle number of the PCR process determined ascharacteristic value for the amplification of the target or standard asn_(T), the product yield of the internal standard or internal controlnucleic acid as Q, the initial amount of internal standard or internalcontrol nucleic acid prior to PCR as Q₀, the efficiency of the PCRamplification with respect to the amplification of the internal standardor internal control as ε_(Q), and the cycle number of the PCR processdetermined as characteristic value for the amplification of the internalstandard or internal control as n_(Q), the difference Δn=n_(Q)−n_(T) canbe used as secondary characteristic value. On basis of the assumptionthat the respective yield T and Q associated to the respective cyclenumber n_(T) and n_(Q) are equal or proportional to each other, thefollowing equation can be derived:log(T ₀ /Q ₀)=log f+Δn log(1+ε)wherein ε=ε_(T)=ε_(Q) has additionally been assumed. This equation canbe used as calibration equation. However, it has been observed that anequation of the formlog(T ₀ /Q ₀)=a Δn ² +b Δn+cmay be more appropriate to describe data obtained in practice.Accordingly this equation or the equationT₀=Q₀ 10^(P)withP=a Δn ² +b Δn+ccan be used advantageously as quantification equation. It may beappropriate to additionally take into account a so-called volume factorv and a so-called recovery factor r, so thatT ₀=(v/r)Q ₀ 10^(P)is obtained.

With reference to one of these calibration equations, the determinationof the calibration data involves basically the determination of therespective equation, in particular of the second order polynomialconstant a, the first order polynomial constant b, the zero orderpolynomial constant c and possibly the volume factor v and the recoveryr (default value for example 1.00). On basis of the measurement resultsand in particular on basis of the determined characteristic values, e.g.the determined secondary characteristic values Δn, the polynomialconstants can be determined by fitting the respective equation to therespective data actually obtained on basis of effecting steps A) to E).This fitting can for example be done by the RMS fitting method. On basisof the respective parameters a, b, c and possibly v and r, in step g)and correspondingly in the respective step of the inventive solutionaccording to the first, more general, aspect, the original amount oftarget nucleic acid in the test sample can be determined using theselected quantification scheme of the first type, with thecharacteristic value for the control nucleic acid and the characteristicvalue for the target nucleic acid or the secondary characteristic valueobtained therefrom (in the example considered here the values n_(T) andn_(Q) or the value Δn) being used as input values.

It should be remarked that as characteristic values for the respectivenucleic acids so-called elbow values can be used. As shown in FIG. 2,the elbow value is the (fractional) cycle number in which the growthcurve is approximately equal to a predetermined threshold value and inwhich exponential growth is still true.

The foregoing makes it sufficiently clear that the amplification processcan comprise a polymerase chain reaction (PCR) process. Further, it hasalready been indicated that the amplification process can comprise or bepart of a reverse transcriptase polymerase chain reaction (RT-PCR)process.

Said target nucleic acid or standard nucleic acid and said controlnucleic acid can be competitively amplified in said amplificationprocess on basis of the same primers for the target nucleic acid orstandard nucleic acid and for said control nucleic acid.

In principle, there are many possible ways that the amplified nucleicacids can be detected. The amplified nucleic acids can be detected bymeans of a detection format that is based on nucleic acid dependentfluorescent signaling. In particular, it can be advantageous if theamplified nucleic acids are detected by means of a detection format thatallows continuous monitoring of the generation of amplificationproducts. For example, the amplified nucleic acids can be detected onbasis of at least one fluorescent-labelled hybridization probe or/and atleast one fluorescent-labelled primer. In one embodiment, afluorescent-labelled hybridization probe is used which is labelled witha fluorescent entity and with a quenching entity, a separation of thefluorescent entity and the quenching entity from each other occurring onhybridization of the hybridization probe to its target sequence, theseparated fluorescent entity being optically excitable to emitfluorescence.

Another possibility is the use of a first fluorescent-labelledhybridization probe and a second fluorescent-labelled hybridizationprobe, the first hybridization probe being labelled with a fluorescentacceptor entity and the second hybridization probe being labelled with afluorescent donor entity, wherein the first and the second hybridizationprobe are adapted to hybridize to neighboring target sequences, andwherein on hybridization to neighboring target sequences the fluorescentacceptor entity is excitable to emit fluorescence via optical excitationof the fluorescent donor entity and fluorescence resonance energytransfer from the fluorescent donor entity to the fluorescent acceptorentity.

Further, one might use a fluorescent-labelled hybridization probe and afluorescent-labelled primer, one thereof being labelled with afluorescent acceptor entity and the other thereof being labelled with afluorescent donor entity, wherein the hybridization probe is adapted tohybridize to a target sequence neighboring to the primer, and wherein onhybridization of the hybridization probe to its target sequence thefluorescent acceptor entity is excitable to emit fluorescence viaoptical excitation of the fluorescent donor entity and fluorescenceresonance energy transfer from the fluorescent donor entity to thefluorescent acceptor entity.

In particular, amplified nucleic acids can be detected on basis of FREThybridization probes, so-called molecular beacons or probes as used inthe TaqMan™ instrument.

It is also possible to detect amplified nucleic adds on basis of aDNA-binding dye, which is optically excitable to emit fluorescence andwhich shows enhanced fluorescence when bound to a double standard DNA.

The method according to the invention can advantageously be performedusing a COBAS TaqMan™ system as provided by Roche Diagnostics.

From the embodiment discussed in the foregoing it is made clear that themethod of the invention can be a method for absolute quantification ofthe respective target nucleic acid. However, it is not ruled out thatthe method is a method for relative quantification of the respectivetarget nucleic acid.

In another implementation, the method of the invention can additionallycomprise the steps of:

-   -   selecting between a quantification of said target nucleic acid        in said test sample and between a determination of presence or        non-presence of said target nucleic acid in said test sample,    -   if selected: determining the presence or non-presence of said        target nucleic acid in said test sample on basis measurement        results obtained in step c), possibly leaving out steps d) to        g).

The invention further provides a quantification kit containing agentsand reference or calibration data to carry out the method of theinvention.

Further, and as shown in FIG. 24, the invention refers to and, accordingto the first, more general, aspect, provides an apparatus 100 forquantification of at least one target nucleic acid in a test sample orin a plurality of test samples in accordance with the method of theinvention. The apparatus according to the invention comprises:

-   -   an amplification unit 102 for effecting a nucleic acid        amplification process with respect to at least one test sample;    -   a detection unit 104 for measuring, at a plurality of different        times during said nucleic acid amplification process effected by        said amplification unit, at least two signals being related to a        respective nucleic acid which is amplified in the amplification        process, the detection mechanism being adapted to independently        measure at least one first signal related only to a first        nucleic acid and at least one second signal related only to a        second nucleic acid or to measure at least one first signal and        at least one second signal from which first data related only to        a first nucleic acid and second data related only to a second        nucleic can be calculated;    -   a controller 106 in communication with said amplification unit        and said detection mechanism;

wherein said controller is adapted or programmed to perform the steps of

-   -   controlling the amplification unit 102 to effect an        amplification with respect to at least one respective test        sample;    -   controlling the detection unit 104 to directly or indirectly        measure the amount of amplification product or a quantity        indicating the amount of amplification product for at least two        different nucleic acids, namely with respect to at least one        first nucleic acid und with respect to at least one second        nucleic acid;    -   determining a characteristic value of said progress parameter        for said first nucleic acid on basis of measurement results        related to the amount of amplification product for said first        nucleic acid;    -   determining a characteristic value of said progress parameter        also for said second nucleic acid on basis of measurement        results related to the amount of amplification product for said        second nucleic acid;    -   quantifying the original amount of target nucleic acid in said        test sample according to a predetermined quantification scheme,        said quantification scheme provides for the quantification of        the original amount of target nucleic acid in said test sample        on basis of the characteristic value for said target nucleic        acid, the characteristic value for said control nucleic acid and        associated reference data;    -   providing quantification data that include said original amount        of first nucleic acid to represent the original amount of target        nucleic in said test sample.

With reference to the above second aspect of the invention, saidcontroller, according to the invention, is adapted or programmed toperform the steps of:

bb) controlling the amplification unit 102 to effect an amplificationwith respect to at least one respective test sample;

cc) controlling the detection unit 104 to directly or indirectly measurethe amount of amplification product or a quantity indicating the amountof amplification product for at least two different nucleic acids,namely with respect to at least one first nucleic acid und with respectto at least one second nucleic acid;

dd) determining a characteristic value of said progress parameter forsaid first nucleic acid on basis of measurement results related to theamount of amplification product for said first nucleic acid;

ee) if certain pre-defined conditions referring to the measured amountof amplification product for said second nucleic acid apply, determininga characteristic value of said progress parameter also for said secondnucleic acid on basis of measurement results related to the amount ofamplification product for said second nucleic acid;

ff) selecting between a plurality of quantification schemes according toat least one predefined selection criterion, said selection beingeffected directly or indirectly on basis of at least one of saidmeasurement results related to the amount of amplification product forsaid first nucleic acid, measurement results related to the amount ofamplification product for said second nucleic acid and saidcharacteristic value or values,

wherein at least one quantification scheme of a first type provides fora quantification of the original amount of first nucleic acid in saidtest sample on basis of the characteristic value for said first nucleicacid, the characteristic value for said second nucleic add andassociated reference data,

wherein at least one quantification scheme of a second type provides fora quantification of the original amount of first nucleic acid in saidtest sample on basis of the characteristic value for said first nucleicacid and associated reference data without reference to anycharacteristic value for said second nucleic acid,

gg) quantifying the original amount of first nucleic acid in said testsample according to the selected quantification scheme on basis of atleast the characteristic value determined for said first nucleic acid,

jj) providing quantification data which include said original amount offirst nucleic acid to represent the original amount of target nucleic insaid test sample.

Said controller further can be adapted or programmed to perform steps b)to j) in accordance with the methods of the invention or/and to performadditional steps in accordance with the methods of the invention.

The invention further refers to and, according to the first, moregeneral, aspect, provides a program of instructions executable by anapparatus 100 for quantification of at least one target nucleic acid ina test sample or in a plurality of test samples, the apparatuscomprising:

-   -   an amplification unit 102 for effecting a nucleic acid        amplification process with respect to at least one test sample;    -   a detection mechanism 104 for measuring, at a plurality of        different times during said nucleic acid amplification process        effected by said amplification unit, at least two signals being        related to a respective nucleic acid which is amplified in the        amplification process, the detection mechanism being adapted to        independently measure at least one first signal related only to        a first nucleic acid and at least one second signal related only        to a second nucleic acid or to measure at least one first signal        and at least one second signal from which first data related        only to a first nucleic acid and second data related only to a        second nucleic can be calculated;    -   a controller 106 in communication with said amplification unit        and said detection mechanism;

wherein said controller in response to said instructions performs thesteps of:

-   -   controlling the amplification unit to effect an amplification        with respect to at least one respective test sample;    -   controlling the detection unit to directly or indirectly measure        the amount of amplification product or a quantity indicating the        amount of amplification product for at least two different        nucleic acids, namely with respect to at least one first nucleic        acid und with respect to at least one second nucleic acid;    -   determining a characteristic value of said progress parameter        for said first nucleic acid on basis of measurement results        related to the amount of amplification product for said first        nucleic acid;    -   determining a characteristic value of said progress parameter        also for said second nucleic acid on basis of measurement        results related to the amount of amplification product for said        second nucleic acid;    -   quantification of the original amount of target nucleic acid in        said test sample according to a predetermined quantification        scheme, said quantification scheme provides for the        quantification of the original amount of target nucleic acid in        said test sample on basis of the characteristic value for said        target nucleic acid, the characteristic value for said control        nucleic acid and associated reference data;    -   providing quantification data that include said original amount        of first nucleic acid to represent the original amount of target        nucleic in said test sample.

With respect to the above second aspect of the invention, saidcontroller in response to said instructions, according to the methods ofthe invention, performs the steps of:

bb) controlling the amplification unit 102 to effect an amplificationwith respect to at least one respective test sample;

cc) controlling the detection unit 106 to directly or indirectly measurethe amount of amplification product or a quantity indicating the amountof amplification product for at least two different nucleic acids,namely with respect to at least one first nucleic acid und with respectto at least one second nucleic acid;

dd) determining a characteristic value of said progress parameter forsaid first nucleic acid on basis of measurement results related to theamount of amplification product for said first nucleic acid;

ee) if certain predefined conditions referring to the measured amount ofamplification product for said second nucleic acid apply, determining acharacteristic value of said progress parameter also for said secondnucleic acid on basis of measurement results related to the amount ofamplification product for said second nucleic acid;

ff) selecting between a plurality of quantification schemes according toat least one predefined selection criterion, said selection beingeffected directly or indirectly on basis of at least one of saidmeasurement results related to the amount of amplification product forsaid first nucleic acid, measurement results related to the amount ofamplification product for said second nucleic acid and saidcharacteristic value or values,

wherein at least one quantification scheme of a first type provides fora quantification of the original amount of first nucleic acid in saidtest sample on basis of the characteristic value for said first nucleicacid, the characteristic value for said second nucleic acid andassociated reference data,

wherein at least one quantification scheme of a second type provides fora quantification of the original amount of first nucleic acid in saidtest sample on basis of the characteristic value for said first nucleicacid and associated reference data without reference to anycharacteristic value for said second nucleic acid,

gg) quantification of the original amount of first nucleic acid in saidtest sample according to the selected quantification scheme on basis ofat least the characteristic value determined for said first nucleicacid,

jj) providing quantification data which include said original amount offirst nucleic acid to represent the original amount of target nucleic insaid test sample.

Said controller 106, in response to said instructions, can perform stepsbb) to jj) in accordance with one of the methods of the invention.

Further, the invention provides a computer program product embodying theprogram. The computer program product can be in the form of a computerreadable medium carrying said program of instructions.

Further, the invention provides a server computer system storing theprogram for downloading via a communication link, possibly via internet.

In the following, exemplary embodiments of the invention are presented.

Some of the illustration examples included herein refer specifically toa COBAS AmpliPrep™/COBAS TaqMan® HBV Test manufactured by RocheMolecular Systems, Inc., USA. However, this particular test serves onlyas an example, and the invention can be applied to any otherquantification test. Accordingly, the following background informationconcerning the HBV Test indicates, for example, how a certainquantification test directed to a particular DNA might be implemented inthe context of the present invention. Of course, other instruments,other amplification mixtures, other detection methods can be used.Accordingly, the COBAS AmpliPrep™/COBAS TaqMan® HBV Test has to beconsidered to be only a non-limiting example which is used herein onlyfor illustration purposes.

The COBAS AmpliPrep™/COBAS TaqMan® HBV Test is an in vitro nucleic acidamplification test for the quantitation of Hepatitis B Virus (HBV) DNAin human serum or plasma, using the COBAS AmpliPrep™ instrument forspecimen processing and the COBAS TaqMan™ Analyzer for amplification anddetection. The test can quantitate HBV DNA over a wide range ofconcentrations. Specimen preparation is automated using the COBASAmpliPrep™ Instrument, and amplification and detection are automatedusing the COBAS TaqMan™ or the COBAS TaqMan 48™ Analyzer.

The COBAS AmpliPrep™/COBAS TaqMan® HBV Test is based on simultaneous PCRamplification of target DNA and detection of cleaved dual-labeledoligonudeotide detection probe that is specific to the target.

The COBAS AmpliPrep™/COBAS TaqMan® HBV Test quantitates HBV viral DNA byutilizing a second target sequence (HBV Quantitation Standard, InternalControl, Internal Standard, IQS, QS) that is added to each test sampleat a known concentration (known copy number). The HBV QuantitationStandard is a non-infectious DNA construct, containing fragments of HBVsequences with primer binding regions identical to those of the HBVtarget sequence. The HBV Quantitation Standard contains HBV primerbinding regions and generates an amplification product of the samelength and base composition as the HBV target DNA. The detection probebinding region of the HBV Quantitation Standard has been modified withrespect to the detection probe binding region of the HBV QuantitationStandard. These unique probe binding regions allow the HBV QuantitationStandard amplicon to be distinguished from HBV. The HBV QuantitationStandard compensates for effects of inhibition and controls thepreparation and amplification processes to allow the accuratequantitation of HBV DNA in each sample.

The COBAS AmpliPrep™/COBAS TaqMan® HBV Test permits automated samplepreparation followed by automated PCR amplification, and detection ofHBV target DNA and HBV Quantitation Standard (Internal Control). TheMaster Mix reagent contains primer pairs and probes specific for bothHBV DNA and HBV Quantitation Standard DNA. The detection of amplifiedDNA is performed using a target-specific and a QuantitationStandard-specific dual labeled oligonucleotide probe that permitsindependent identification of HBV amplicon and HBV Quantitation Standardamplicon. The COBAS TaqMan™ Analyzer calculates the HBV DNAconcentration in the test samples by comparing the HBV signal to the HBVQuantitation Standard signal for each sample and control.

Processed samples are added to the amplification mixture inamplification tubes (K-tubes) in which PCR amplification occurs. TheThermal Cycler in the COBAS TaqMan™/TaqMan 48™ Analyzer heats thereaction mixture to denature the double stranded DNAs and expose thespecific primer target sequences on the HBV circular DNA genome and theHBV Quantitation Standard DNA. As the mixture cools, the primers annealto the respective target DNA sequence of the HBV target DNA and to theHBV Quantitation Standard DNA. Under appropriate conditions the DNApolymerase extends the annealed primers along the target template toproduce double-stranded DNA molecule termed an amplicon.

The COBAS TaqMan™/TaqMan 48™ Analyzer automatically repeats this processfor a designated number of cycles, with each cycle intended to doublethe amount of amplicon DNA. The required number of cycles ispreprogrammed into the COBAS TaqMan™/TaqMan 48™ Analyzer. Amplificationoccurs only in the region of the HBV genome between the primers; theentire HBV genome is not amplified.

The COBAS AmpliPrep™/COBAS TaqMan® HBV Test utilizes state of the artPCR technology, including for example the probes and detection formatsused in the TaqMan™ instrument. The use of dual-labelled fluorescentprobes allows for real-time detection of PCR product accumulation bymonitoring of the emission intensity of fluorescent reporter dyesreleased during the amplification process. The probes consist of an HBVand HBV Quantitation Standard-specific oligonucleotide probes with areporter dye and a quencher dye. In the COBAS AmpliPrep™/COBAS TaqMan®HBV Test, the HBV and HBV Quantitation Standard probes are labeled withdifferent fluorescent reporter dyes. When the probes are intact, thereporter fluorescence is suppressed by the proximity of the quencher dyedue to Förster-type energy transfer effects. During PCR, the probehybridizes to a target sequence and is cleaved by the 5′->3′ nucleaseactivity of the DNA polymerase. Once the reporter and quencher dyes arereleased and separated, quenching no longer occurs, and the fluorescentactivity of the reporter dye is increased. The amplification of HBV DNAand HBV Quantitation Standard DNA are measured independently atdifferent wavelengths. During the annealing phase of the PCR on theCOBAS TaqMan™ Analyzer, the samples are illuminated and excited byfiltered light and filtered emission fluorescence data are collected foreach sample. This process is repeated for a designated number of cycles,each cycle effectively increasing the emission intensity of theindividual reporter dyes, permitting independent identification of HBVand HBV Quantitation Standard DNA. The PCR cycle where a growth curvestarts exponential growth is related to the amount of starting materialat the beginning of the PCR.

The PCR used is quantitative over a wide dynamic range since themonitoring of amplicon is performed during the exponential phase ofgrowth. The higher the HBV concentration of a sample, the earlier thefluorescence of the reporter dye of the HBV probe rises above thebaseline fluorescence level. Since the amount of HBV QuantitationStandard (QS) DNA is constant between all samples, the fluorescence ofthe reporter dye of the HBV QS probe should appear at or near the samecycle for all samples. In case where the QS fluorescence is affected,the concentration is adjusted accordingly. The appearance of a specificfluorescent signal is reported as a critical threshold value (ct), andin the following is also denoted as an “elbow value” nT or ct_(T),referring to the target (in the present example the HBV target DNA) andas an “elbow value” nQ or ct_(QS), referring to the internal control (inthe present example the HBV quantification standard DNA). The ct isdefined as the fractional cycle number where reporter dye fluorescenceexceeds a predetermined threshold, and starts the beginning of anexponential growth phase of this signal. A higher ct value indicates alower concentration of initial target material

Target growth curves for a dilution series spanning a desired range canbe obtained. As shown in FIGS. 13A, 13C, 15A and 15C, as theconcentration of the sample increases the growth curves shift to earliercycles. Therefore the leftmost growth curve corresponds to the highesttarget concentration level whereas the rightmost growth curvecorresponds to the lowest target concentration level. For each growthcurve the fluorescence values at every cycle are normalized. Thefractional ct value is calculated where the fluorescence signal crossesa predefined fluorescence level.

Lot-specific calibration constants provided with the COBASAmpliPrep™/COBAS TaqMan® HBV Test are used to calculate theconcentration value for the initial sample based upon the HBV DNA andHBV Quantitation Standard DNA ct values.

As exemplified in the COBAS AmpliPrep™/COBAS TaqMan® HBV Test,differences between the ct values of the HBV DNA and HBV quantificationstandard DNA can be calculated, and the concentration value for theinitial sample can be calculated on basis of these difference values andthe lot-specific calibration constants.

According to one embodiment, the determination of the lot-specificcalibration constants is considered part of the method provided by theinvention. The calibration constants can be determined by quantitating adilution series of target DNA (e.g. HBV DNA), wherein each sample of thedilution series includes the same known concentration of the internalcontrol (e.g. HBV quantitation standard) which is also included in therespective test (e.g. COBAS AmpliPrep™/COBAS TaqMan® HBV Test).According to the terminology used in the field, this calibration iseffected on basis of an “external standard”.

It should be added that it is advantageous but generally not necessarythat the internal control (internal quantification standard) on the onehand and the target (see above example of HBV viral DNA) are similarwith identical primer regions and amplification products of the samelength and base composition. One might even use different primers.However, using similar target and internal control sequences andidentical primers has the advantage that the amplification efficienciesfor both amplification reactions occurring simultaneously should beidentical, or nearly identical.

In the following, examples referring to the calibration of a respectivequantification test and the calibration part of a corresponding methodprovided by the invention are presented.

The calibration of a given quantification test which uses an internalcontrol can, according to one embodiment of the invention, be effectedfor example as illustrated in FIGS. 1 a and 1 b. In a first step (stepA-1) a dilution series of samples is provided which each include arespective copy number T_(0i) of the target nucleic acid (referring tothe above example e.g. HBV DNA) and the same number of internal controlor quantification standard nucleic acid (referring to the above examplee.g. HBV quantification standard DNA constructs). The dilution seriesincludes S individual samples.

Using appropriate instrumentation, e.g. the COBAS TaqMan™ Analyzer, thesamples are amplified by PCR-amplification over P cycles (step A-2).During the amplification, the fluorescence intensities indicating thePCR product accumulation are measured. This can be done on basis ofprobes and detection formats as used in the TaqMan™ instrument or onbasis of other means known in the art. There can be crosstalk betweenfluorescence signals due to an overlap of the fluorescence emissionspectra and receiving bandwidth of the fluorescence detector associatedto another PCR product. This crosstalk can be corrected by effecting amulticomponent analysis. For example, a so-called crosstalk calibrationon basis of color standards can be effected to provide an essay-specificcrosstalk matrix which can be used to convert a filter-reading vectorobtained from the measurements into a crosstalk-free fluorescentintensity vector.

On basis of the measured fluorescence intensities for each sample atarget growth curve T and a control growth curve Q are obtained (stepA-3).

If desired, pre-checks and corrections can be effected with respect tothe growth curves, for example to eliminate artifacts, to compensate forinstrumental fluctuations and the like. The growth curves can benormalized, for example by dividing each growth curve raw fluorescencevalue by the intercept value of the growth curve base line with theordinate. FIG. 2 shows a corresponding example.

On basis of the growth curves, for example, on basis of the normalizedgrowth curves, a respective characteristic value indicating a certainmomentarily status of the amplification is determined (step A-4). In thepresent example, so-called “elbow values” nT for the target growthcurves and nQ for the control growth curves are determined whichcorrespond to a fractional cycle number at which the respective growthcurve crosses an arbitrary signal value ASV (FIG. 2). To obtain afractional cycle number an appropriate interpolation of the growth curvebetween growth curve points obtained from the measurement can beeffected.

Generally, for each sample i a target elbow value nT_(i) and a controlelbow value nQ_(i) is obtained (step A-5). However, for high initialtarget concentrations T₀ it might happen that the PCR amplification withrespect to the internal control gives no characteristic growth curvewithin the cycles 1 to P, so that no control elbow value can beobtained. Such a dropout of the control growth curve is caused bycompetitive effects between the target and the internal control. Themethod might be implemented appropriately to take into account possibledropouts of the control growth curves. In the following steps only thosesamples should be taken into account for which appropriate target elbowvalues nC_(i) and appropriate control elbow values nQ_(i) were obtained.Corresponding checks of the elbow values, which were determined, can beimplemented on basis of theoretical considerations, in particular thedynamics of the PCR amplification.

For all appropriate pairs nT_(i), nQ_(i) a respective elbow differenceΔn_(i)=nQ_(i)−nT_(i) is calculated (step A-6). Accordingly, for eachsample of the dilution series or, in case of control growth curvedropout, for each sample of a subset of the dilution series a value pair(T_(0i), Δn_(i)) is obtained (step A-7) which relates the respectiveinitial target concentration T_(0i) to the elbow difference Δn_(i)determined for the respective sample i.

These value pairs allow the calibration of the respective quantificationtest (step A-8). For example, a theoretical calibration formula derivedfrom the exponential amplification function can be used. According tothe example considered here, a quantification formula (quantitationequation) of second order is used which was shown to accuratelyrepresent the relationships between the target and control growthcurves. This second order polynomial equation can sufficiently take intoaccount competitive effects in the co-amplification (e.g. primercompetition). By fitting the calibration formula to the data pairs(T_(0i), Δn_(i)) for example by the RMS method or the KRC method, theappropriate parameters a, b and c of the calibration formula shown inFIG. 1 b can be obtained, which serve as calibration constants. Further,the amplification efficiency ε and a proportionality constant f betweenthe amplification of the target and the amplification of the internalcontrol can be obtained.

The calibration can be further illustrated on basis of numericalexamples shown in FIGS. 3 to 8. FIG. 3 is a sheet of input data andamplification result data for 24 samples. Before the PCR amplification,each sample included the same copy number of internal control nucleicacids (1000 copies). In the second column the initial amount (copynumber) of target nucleic acid for the respective sample is given.Columns 4 and 5 show the elbow value n_(Qs) of the amplification of theinternal control and the elbow value n_(T) of the PCR amplification ofthe target. The difference between these elbow values is given in column6.

FIG. 4 is a plot of the log(target input copy #/IQS input copy #) versus(elbow of IQS—elbow of target). Further, two calibration curves, one offirst order and the other of second order, are plotted as fitted to thedata points obtained from the amplification. For the fit, the KRC methodwas used. The slope and intercept of the linear fit according to the KRCmethod yields the co-amplification efficiency ε and the constant ofproportionality f.

To estimate the degree of fit one can back-calculate the titers usingthe regression data. FIG. 5 shows, in comparison with the respectiveactual target input copy number, such back calculations on basis of thelinear and polynomial regression data, as well as on basis of theexponential amplification formula using values for f and ε obtained fromthe fit.

It is possible to additionally fit the target elbow values (reporterelbow values) on the one hand and the internal control (IQS) elbowvalues on the other hand independently of each other to respectivecalibration formula. Corresponding examples are shown in FIGS. 6 and 7.According to one embodiment of the method, such calibration data areused for the quantification of samples which show a dropout of thecontrol growth curve or a degenerated control growth curve, so that noappropriate control elbow value or no control elbow value at all couldbe determined.

FIG. 8 shows the back calculation results on basis of the regressionfits correlated to the actual data.

It has already been mentioned that for quantification of samples havinghigh initial target concentrations an additional calibration can beeffected. It is referred to FIG. 9. According to a first variant denotedas B1 the initial target copy numbers (or concentrations) T_(0i) and therespective target elbow values nT_(i) of the samples used for thecalibration according to FIGS. 1 a and 1 b are accessed (step B2-2) andused (step B1-2) to determine calibration constants A, B, C on basis ofan appropriate calibration formula, e.g. second order polynomialequation. Since the control elbow values are not needed, theamplification results for all samples can be used, even if there havebeen control growth curve dropouts. Beside the calibration constants,the amplification efficiency can be provided (step B1-3) on basis of acorresponding fit of the calibration formula to the data points.

According to a more simple approach, only the amplification result for aselected sample j is accessed (step B2) and used which has an elbowvalue nT_(j) corresponding approximately to a predefined reference valuenT_(ref). The predefined reference value nT_(ref) corresponds to arelatively high initial target concentration (copy number), leading to arelatively low elbow value, so that for this target concentration or forsomewhat higher target concentrations there is a higher probability ofcontrol growth curve dropouts. The value pair (T_(0j), nT_(j)) itselfcan be used as calibration constant on basis of the exponentialamplification function, possibly together with a theoreticalamplification efficiency or with the amplification efficiency obtainedfrom the calibration referred to in the foregoing.

It should be added that, in agreement with the examples discussed in theforegoing, elbow values or threshold values are determined on basis ofthe growth curves. However, instead of such elbow or threshold valuesone may use other characteristic values, for example determined on basisof the first, second or nth derivatives of a respective growth curve.

With reference to FIG. 10 a and FIG. 10 b the quantification of samplescontaining an unknown target titer T₀ can be effected as follows. Afterproviding one sample or a set of samples with unknown target titerT_(0i) and equal known control titer Q₀ as used in the calibration (stepC-1), the PCR amplification with measurement of the fluorescenceintensity is effected as in the method part referring to the calibration(step C-2). If necessary, crosstalk correction is effected. From theamplification a respective target growth curve T_(i) is obtained andgenerally also a control growth curve Q_(i) (step C-3). After effectingpre-checks and corrections (if desired), the data are normalized andelbow values for the growth curves are determined (step C-4). Generally,for each sample a target elbow value nT_(i) and a control elbow valuenQ_(i) are obtained (step C-5). However, there might be a dropout of thecontrol growth curve, if the initial target titer T₀ was relativelyhigh. For such samples, only a target elbow value nT_(i) is obtained.However, there is also the possibility that control elbow values nQ_(i)occur which are not appropriate and have to be discarded.

After calculating the elbow differences Δn_(i) (step C-6), the inputvalues Δn_(i) for the calibration formula are obtained (step C-7). Onbasis of the calibration formula and the constants a, b, c obtained fromthe calibration the initial titer values T_(0i) can be calculated (stepsC-8, C-9).

For samples which showed a dropout in the control growth curve, so thatno control elbow value could be determined, or for which aninappropriate control elbow value was obtained, the initial titer T_(0i)can be obtained on basis of the respective target elbow value nT_(i)using an appropriate calibration formula and associated calibrationdata, e.g. the formula and data according to embodiments B1) and B2) ofFIG. 9. It is referred in this respect to the process according to stepsD1-1, D1-2 and D1-3 and according to steps D2-1, D2-2 and D2-3 asillustrated in FIG. 11 a and FIG. 11 b.

In the following, the invention is further exemplified on basis of aparticular example, namely the COBAS AmpliPrep™/COBAS TaqMan® HBV Testreferred to in the foregoing. FIG. 12 shows the print on the packet ofthe COBAS CAP/CTM HBV Test kit used. This test kit will be obtainablefrom Roche Molecular Systems, Inc., Branchburg, N.J. USA, or from RocheDiagnostics GmbH, Mannheim, Germany.

FIG. 13 shows growth curves for a dilution series of HBV plasmid targetand internal controls obtained on basis of the HBV test according toFIG. 12. The dilution series of HBV plasmid is a series of 1e1 to 1e9CTMU/ml target sequence and 5.0 e3 CTMU/ml internal control sequence.The unit CTMU refers to a so-called COBAS TaqMan™ unit. There is acalibration factor generally between 1 and 3 relating 1 CTMU/ml to acertain copy number/ml. In view of the merely illustrative purposesherein one might identify one CTMU as one copy for simplicity.

To the solution series two samples of WHO-EUROHEP-Standard including 1e4CTMU/ml target were added for reference purposes.

FIG. 13 a shows the unnormalized target growth curves, and FIG. 13 bshows the unnormalized control growth curves. A normalization whichremoves the baseline offset leads to the target growth curves of FIG. 13c and the control growth curves of FIG. 13 d.

In the control growth curves, there is no internal control failure value(no dropout of a control growth curve) visible. Even for the highestconcentrations of HBV plasmid (1e8 and 1e9) the growth curves of theinternal control show a distinct growth and separate distinct from thebaseline. Accordingly, a desired titer measurement range of for exampleabout 5 decades is reached. In principle, it is sufficient to use thestandard quantification on basis of the elbow differences. FIG. 14indicates respective elbow values for the target growth curves. Theelbow values for the control growth curves are not shown.

The situation is different for another target dilution series rangingfrom 5e2 CTMU/ml to 1e9 CTMU/ml of target HBV plasmid, with 5.0 e3CTMU/ml internal control, for which the unnormalized and normalizedgrowth curves are shown in FIG. 15. In this dilution series theWHO-EUROHEP-Standard of 1e4 CTMU/ml was added (two samples). Althoughthe target curves still show a nice and smooth dependence and reflectthe concentrations as expected, the internal control suffers obviouslyfrom high target concentrations. At concentrations of 1e8 and highervery low internal control signals were obtained, so that the probabilityof a complete failure of the standard quantification on basis of elbowdifferences is dramatically increased. For some samples obviously nocontrol elbow value can be determined, so that the standardquantification fails. FIG. 16 indicates respective elbow values for thetarget growth curves. The elbow values for the control growth curves arenot shown.

In the following, embodiments and modifications of the calibration andquantification considered on basis of FIGS. 1 to 11 are presented.Formula F0 of FIG. 17 a shows the standard titer calculation formula forCOBAS TaqMan™ instrument growth curves using elbow value differences.Formula F0 can be written in a simplified format, with constants a, b, cfor a fixed internal control copy number Q, for a fixed recovery r andfor a fixed volume factor v, since it is not necessary to treat Q, r andv as parameters. Accordingly, the standard calibration formula alreadyconsidered in the foregoing is obtained.

To avoid the use of inappropriate control elbow values, one maycalculate the target titer directly from the elbow value ct_(T) (denotedas nT in the foregoing) of the target growth curve, if the elbow valueof the target is below a threshold value of for example 20. For the HBVtest a target elbow value of 20 corresponds to about 5*10⁶ copies. Forsuch relatively small target elbow values, formula F1 can be used inagreement with the “exceptional” quantification according to steps D2-1,D2-2 and D2-3. The high titer calculation function F1 assumes anexponential growth with amplification factor AF=1+ε=2 per cycle. This isa somewhat idealistic assumption. It must be expected, that thisapproach generally overestimates the real growth and that a correctionto an amplification factor smaller than 2 would improve the quality ofhigh titer quantitation.

The values of T_(ref) and ct_(ref) (denoted as nQ in the foregoing) arereference values which are determined in a calibration procedure, forexample on several instruments, for example, a high titer calibrationB2. These values can be determined with respect to each reagent kit lotof a certain test. Within the calibration, basically two steps can beperformed: i) quantification of the control reagent with reasonableposition and accuracy and assignment to T_(ref), ii) determination of anaverage ct which is found if the control reagent is applied to differentinstruments and assignment to ct_(ref). The values for T_(ref) andct_(ref) can be provided together with a respective reagent kit. Forexample, these values can be coded in bar code on reagent cassettes.When the reagent cassettes are loaded onto an amplification instrument(e.g. COBAS AmpliPrep™ Instrument) the T_(ref) and ct_(ref) data can beread by a bar code detector and transferred as input data to the controlsoftware of the instrument. Depending on the accuracy needed and theuniformity of the reagent kit lots lot specificity might not benecessary, so that it would be sufficient to determine T_(ref) andct_(ref) test specifically.

One particular issue is that T_(ref) should be found in a certain “ctrange” (e.g. 19.3<ct_(ref)<20.7). This corresponds to a log error of±0.21. Further, T_(ref) must be found in that “ct range” for allinstruments. An alternative to this approach is the approach accordingto steps B1-1, B1-2 and B1-3 (calibration) and steps D1-1, D1-2 and D1-3(quantification), using a second order calibration formula.

Instead of using a fixed theoretical amplification factor, one might useinstead an amplification factor AF=1+ε per PCR circle which is derivedfrom the calibration curve or data of the standard calibration. Acorresponding formula is denoted as F2.

By looking at the standard calibration formula, the amplification factorAF is obtained for Δct=ct_(QS)−ct_(T)=0 (AF=e^(b*ln10b); b being thesecond parameter of the calibration coefficients). Accordingly, by usingAF=e^(b*ln10) instead of AF=2 in formula F2 it is possible to extractthe amplification factor from the calibrated set of data and to use thisfactor also in the high target concentration region.

FIG. 18 shows a flow chart part of a complete titer calculationalgorithm in which, dependent on the value of the target elbow valuect_(T) with respect to the reference elbow value ct_(ref), either thestandard quantification formula or the “exceptional” quantificationformula F1 (or alternatively the “exceptional” quantification formulaF2) is used.

A modification of this flow chart part is shown in FIG. 19. There aretwo conditions, which must be fulfilled for the exceptionalquantification formula F1 or F2 to be applied. The additional conditionis that the normalized internal control intensity value afi(n) after thelast PCR amplification cycle does not exceed a certain threshold valueafi(n)_(ref). The threshold value afi(n)_(ref) must be selectedappropriately with respect to the normalization effected. For thenormalizations shown in FIGS. 13 to 16 a threshold value afi(n)_(ref)<2means a very low plateau for the internal control QS. If these twoconditions hold, the target titer is calculated directly from the ct ofthe target growth curve by using for example formula F1 or formula F2.

The modification according to FIG. 19 means that the exceptionalquantification formula is only applied when the internal control curvefails completely or if this curve becomes very weak with a plateau valuesmaller than the threshold value. Accordingly, for growth curves asshown in FIG. 13 still the standard quantification formula would be usedwith respect to all samples.

An evaluation of the growth curves according to FIGS. 13 to 16 on basisof the standard quantification formula, the exceptional quantificationformula F1, and the exceptional quantification formula F2 leads to theback calculation results of FIGS. 20 and 22 and the signal recoveryresults of FIGS. 21 and 23. Four quantification approaches were applied.The approach “standard” is based on the standard quantification formulaonly. The approach “F1” is based on the standard calibration formula andthe exceptional quantification formula F1 together with the relevantflow chart part of FIG. 18. The approach “F2” is based on the standardquantification formula and the exceptional quantification formula F2,again in combination with the relevant flow chart part of FIG. 18.Further, the approach “F3” is again based on the standard quantificationformula and the exceptional quantification formula F1, but not incombination with the relevant flow chart part shown in FIG. 18. Instead,the relevant flow chart part shown in FIG. 19 was used.

FIGS. 20 and 21 were obtained for the growth curve of FIG. 13. There areonly small deviations between the quantification results obtained forthe different approaches. The standard titer calculation works fine forthe shown data because there is no failure in the internal controlgrowth curves up to highest target concentrations. Further, the approach“F1” using the exceptional formula F1 in combination with the conditionof FIG. 18 gives very good results. The calculation of all target yieldsabove˜4e6 shows no visible deviation from the standard function. Indeed,it appears that the high yield values do not scatter as much as thestandard function. This is reasonable because one can expect that theinternal control signal shows an increased scatter at high targetyields. This scatter cannot contribute in quantification formula F1.

Also, the exceptional quantification formula F2 used in combination withthe condition of FIG. 18 (approach “F2”) describes the titerexcellently, like formula F1. The AF factor derived from the calibrationcurve (for the presented data: 1.95) is adequate for the high titerrange.

Approach “F3” (formula F1 in combination with the condition of FIG. 19)gives the same results as the standard quantification formula since forthe samples the afi(n) values of the internal control (QS) growth curveexceed the preset threshold value afi(n)_(ref)=2. Accordingly, theexceptional quantification formula was never applied.

The signal recoveries shown in FIG. 21 indicate, that a specificationlimit of log error ±0.3 represented by bars is not attained over thewhole dynamical range and for all formulas. However, on the whole,reasonable performance is obtained, in particular for the standardquantification formula and formula F1.

FIGS. 22 and 23 are based on the amplification results of FIG. 15.Again, the approaches “standard”, “F1”, “F2”, and “F3” as defined withrespect to FIGS. 20 and 21 were used.

The standard quantification formula of the approach “standard” cannotderive a target titer if the internal control growth curve fails. Due tothis there are no data points at the highest input copy number (1e9) andonly two determinations, but quite off the real value, for theconcentration 1e8.

At lower target concentrations (in the evaluation shown below 4e6) thestandard quantification equation is used according to approach F1.Differentiation between the exceptional quantification formula F1 andthe standard quantification formula is visible at target yields>4e6. Dueto the failures of the internal control at concentrations 1e8 and 1e9the exceptional quantification according to approaches F1 and F3 givesuperimposing results at these yields. Although quantification formulaF1 is a very simple function and the assumption AF=2 is quiteidealistic, this formula delivers excellent results for this set ofdata.

Quantification formula F2 according to approach “F2” with theamplification factor AF=e^(b*ln10) overestimates the titer considerably.The amplification factor AF derived from the standard calibration curveappears to be not adequate (too high) for the high titer range.Accordingly, it might be appropriate to implement additional pre-checksto avoid an overestimation on basis of amplification factors derivedfrom the calibration curve which are too high in view of the theoreticaldynamics of the PCR amplification reaction. It might be appropriate tocombine the amplification factor derived from the calibration curve witha predetermined theoretical amplification factor appropriately, forexample by taking always the lower of the two amplification factors.

Approach “F3” on basis of exceptional quantification formula F1 and thecondition of FIG. 19 give excellent results over the whole measurementrange.

FIG. 23, as did FIG. 21, illustrates the deviation of the expectedversus the found copy numbers on a log scale. The bars represent anassumed maximum deviation limit (±0.3 log). At low target titers thedata points of the different functions are equal. Starting at 1e7 thefunctions separate from each other. At 1e8 only two values from thestandard quantification approach appear which deviate substantially fromthe expected value. The presence of rectangles at log value “−3.0” comefrom all titer values which could not be determined due to failure ofthe internal control growth curves on basis of the standardquantification approach.

FIG. 24 shows schematically an example for the structure of a system,which can be used for implementing the invention. The system orquantification apparatus 100 has an amplification unit 102 which isadapted to perform PCR-amplification. In particular, a thermal cycler asknown from EP 0 955 097 A1 can be included. Further, there is adetecting unit 104 for measuring fluorescent light emitted bysample-reagent mixtures amplified within amplification unit 102. Devicesas known from EP 0 953 379 A1 and EP 0 953 837 A1 can be provided.

Both units 102 and 104 are controlled by a control unit 106, for examplea work station on basis of an industry standard operating system and anindustry standard micro processor. In particular, the control unitcontrols the amplification cycles effected by the amplification unit 102and receives fluorescence intensity data from the detection unit 104. Acontrol program defining the control processing and the data processingeffected by the control unit 106 can be stored in a storage unit 108,e.g. a magnetic disc unit. Of course, there is also sufficient RAMmemory. An input unit 110 is provided for inputting input data including(if desired) external calibration data into the system. The input unitcan comprise a keyboard, a floppy disc drive or CD-ROM drive and abarcode reader, to give some examples. Further, there is an output unit112, for example, comprising a display and a printer which serve inparticular for outputting the quantification results.

In particular, for performing the method according to the invention aconventional Roche COBAS AmpliPrep™/COBAS TaqMan™ system as provided byRoche can be used, which, however, was adapted to perform the inventionby providing control software which controls the system and interactswith the components of the system in such a way that the method of theinvention is performed. In particular, the flow chart parts shown inFIGS. 18 and 19 and the alternate calibration formulas shown in FIGS. 17a and 17 b can be implemented by the person skilled in the art withoutany problem in the control software of such a system using conventionalprogramming techniques. Referring to FIGS. 1 a, 1 b, FIGS. 9, 10 a, 10 band FIGS. 11 a, 11 b it should be remarked that these figures can beseen as diagrams indicating the data flow occurring in such a system andthe control steps and data processing effected within such a system. Onbasis of the information contained herein the person skilled in the artis enabled to adapt a conventional quantification apparatus or thesoftware thereof to perform the invention. Further, the person skilledin the art is enabled to provide software implementing the invention,which, when loaded in a conventional system, transforms the conventionalsystem in a system embodying the invention.

Some specific embodiments of the invention referred to in the foregoingmay be described as follows:

i) Quantification of Samples:

-   -   The samples are commonly amplified with an internal control by        means of PCR.    -   Real time PCR is used (no end-point method).    -   The amplification products are detected by detection technology        using two different fluorescent labels for the target and the        internal control, e.g. in agreement with the detection formats        and probes of the TaqMan™ instrument.    -   Threshold values or elbow values are determined on basis of the        growth curves by defining a fixed threshold. The first, second        or nth derivative of the growth curve is not used (however, this        is possible in principle).    -   The fractional cycle numbers, at which the growth curve of the        target and the growth curve of the internal control reach the        threshold, are estimated.    -   From the difference between these fractional cycle numbers the        initial titer of the target can be derived on basis of a        predefined calibration formula or calibration curve.

ii) Providing the Calibration Formula or Calibration Curve:

-   -   A calibration panel with several dilution steps (a dilution        series), which for example is adjusted on basis of the        WHO-EUROHEP-Standard, is amplified on basis of several replica        nucleic acids by means of state of the art PCR, e.g. in        agreement with the detection formats and probes of the TaqMan™        instrument. The threshold cycles are determined. For each        dilution stage (standard) the internal control is coprocessed in        a fixed predetermined concentration.    -   The cycle differences between the target and the internal        control which are determined for the respective standard are        determined and evaluated on basis of a calibration function or        formula. E.g., said cycle differences and the respective initial        titer of the standards can be plotted in a diagram (cycle        differences versus initial target titer).    -   An associated calibration curve or/and a calibration function        can be generated which enable to calculate or determine the        initial titer for arbitrary cycle differences which are        measured.

iii) Quantification of Samples on Basis of a Bimodal or MultimodalQuantification Scheme:

-   -   The samples are commonly amplified with an internal control by        means of PCR.    -   Real time PCR is used (no end-point method).    -   The amplification products are detected by detection technology        using two different fluorescent labels for the target and the        internal control, e.g. in agreement with the detection formats        and probes of the TaqMan™ instrument.    -   Threshold values or elbow values are determined on basis of the        growth curves by defining a fixed threshold. The first, second        or n. derivative of the growth curve is not used (however, this        is possible in principle).    -   The fractional cycle numbers, at which the growth curve of the        target and the growth curve of the internal control reach the        threshold, as estimated.    -   Depending on the situation, a standard quantification or an        alternative (exceptional) quantification is selected. According        to a first approach, an alternative quantification of the titer        is selected, if the growth curve of the target reaches the        threshold for a very early cycle, to evaluate the titer without        reference to the cycle associated to the internal control.        According to a second approach, there is the additional        condition, that the alternative quantification formula is only        selected, if the internal control concentration at the final        stage of the amplification does not exceed a minimum plateau        value. Generally, an alternative quantification of the target        titer might be selected when the internal control drops out or        is impaired due to competition, as can be expected for high        target concentrations.

According to these approaches the alternative quantification of theinternal control signal is sample-selectively not included in thecalculation of the target titer for high target concentrations.

-   -   In other cases the initial target titer is evaluated on basis of        the determined cycle differences using a predefined calibration        curve or calibration formula (standard quantification).

iv) Providing Calibration Data for the Bimodal or MultimodalQuantification Scheme:

-   -   A calibration panel with several dilution steps (a dilution        series), which for example is adjusted on basis of the        WHO-EUROHEP-Standard, is amplified on basis of several replica        nucleic acids by means of state of the art PCR, e.g. in        agreement with the detection formats and probes of the TaqMan™        instrument. The threshold cycles are determined. For each        dilution stage (standard) the internal control is coprocessed in        a fixed predetermined concentration.    -   The cycle differences between the target and the internal        control which are determined for the respective standard are        determined and evaluated on basis of a calibration function or        formula. E.G., said cycle differences and the respective initial        titer of the standards can be plotted in a diagram (cycle        differences versus initial target titer).    -   An associated calibration curve or/and a calibration function        (generally calibration data) can be generated which enable to        calculate or determine the initial titer for arbitrary cycle        differences which are measured.    -   For the high titer range a second calibration curve or/and        calibration function (generally calibration data) is generated        on basis of the initial standard titer(s) (target) and the        detected threshold cycle(s).    -   For arbitrary cycle differences measured or—in case of high        initial target concentration—for arbitrary target threshold        cycles the respective initial titer can be calculated or        determined on basis of the calibration data (e.g. two        calibration curves or formulas).

The above examples and experimental results show that it is possible tocircumvent the problem of failure of the internal control at high titerconcentrations of the target by providing at least two quantificationschemes of different type, one of them using the characteristic valuefor the target nucleic acid as well as the characteristic value for thecontrol nucleic acid and another using only the characteristic value forthe target nucleic acid for the quantification. In particular, theinvention provides a bimodal or multimodal titer calculation algorithm,for which alternative quantification approaches (e.g. quantificationcalibration formulas) might be used.

The benefits of the bimodal or multimodal quantification are inparticular the following:

-   -   It is possible to determine titers with specified precision up        to very high yields (>10¹⁰ cps), which extends the dynamic range        of conventional nucleic acid testing considerably.    -   High target concentrations have generally very solid and robust        growth curves. The precision and recovery of these specimen        would suffer considerably from the variance of less solid and        less robust internal control growth curves. Thus, if the titer        calculation for such high target considerations is done without        internal control the quality of the quantification results can        be improved (lower coefficient of variation (CV)).    -   Dropouts of the internal control signal which are caused by        competition between the target and the internal control growth        can appropriately dealt with. For practical applications, the        dropout or generally the use of an “exceptional calibration        formula” could be signaled to the user appropriately, so that        the user is aware, that the internal control functionality of        the internal control (IQS) was not evaluated (i.e. for high        target concentrations when the internal control signal becomes        very low or falls out completely).    -   It is not necessary to adjust the applied internal control copy        number to higher values to enable an extended dynamic range to        upper values of initial target titer. Instead it is possible to        keep the internal control copy number low so that the        competitional behavior does not effect the sensitivity of the        assay.    -   For practical application the switching point between a high        titer quantification function (“exceptional” calibration        formula) and the standard evaluation (standard calibration        formula) can be adjusted appropriately, depending on the desired        dynamic range and the applied internal control copy number.    -   As far as a theoretical amplification factor is used, it might        be appropriate to define the amplification factor test- or        kitlot-specific. Compared to the theoretical value AF=2 used in        foregoing, it might be appropriate to adjust the factor slightly        to smaller values.    -   The use of a titer calculation formula without reference to the        internal control for high initial titer values appears to be        even more appropriate since the accuracy of a titer measurement        scales inversely with the number of PCR cycles. Accordingly, at        high target yields the error is smaller, because less cycles are        involved in the quantification process. Accordingly, the        internal control functionality of the internal control is less        important.

The invention can be implemented in digital electronic circuitry, or incomputer hardware, firmware, software, or in combinations of them.Apparatus of the invention can be implemented in a computer programproduct tangibly embodied in a machine readable storage device forexecution by a programmable processor; and method steps of the inventioncan be performed by a programmable processor executing a program ofinstructions to perform functions of the invention by operating on inputdata and generating output.

The invention can be implemented advantageously in one or more computerprograms that are executable on a programmable system including at leastone programmable processor coupled to receive data and instructionsfrom, and to transmit data and instructions to, a data storage system,at least one input device, and at least one output device. Each computerprogram can be implemented in a high level procedural or object orientedprogramming language, or in assembly or machine language if desired; andin any case, the language can be a compiled or interpreted language.Suitable processors include, by way of example, both general and specialpurpose microprocessors. Generally, a processor will receiveinstructions and data from a read only memory and/or a random accessmemory. The essential elements of a computer are a processor forexecuting instructions and a memory.

Generally, a computer will include one or more mass storage devices forstoring data files; such devices include magnetic disks, such asinternal hard disks and removable disks; magneto-optical disks; andoptical disks. Storage devices suitable for tangibly embodying computerprogram instructions and data include all forms of non-volatile memory,including by way of example semiconductor memory devices, such as EPROM,EEPROM, and flash memory devices; magnetic disks such as internal harddisks and removable disks; magneto-optical disks; and CD-ROM disks. Toprovide for interaction with a user, the invention can be implemented ona computer system having a display device such as a monitor or LCDscreen for displaying information to the user and a keyboard and apointing device such as a mouse or a trackball by which the user canprovide input to the computer system.

The invention was illustrated in the foregoing on basis of a number ofexemplary embodiments and illustrative examples and measurement results.Other embodiments are possible. Accordingly, the invention shall only belimited by the contents and scope of the attached claims.

1. A method for quantification of at least one target nucleic acid in atest sample, the method comprising: a) providing at least one targetnucleic acid together with at least one internal control in a commontest sample, said internal control comprising a defined initial amount(Q₀) of a control nucleic acid different from said target nucleic acid;b) amplifying said target nucleic acid and said control nucleic acidwithin said test sample in a common nucleic acid amplification process;c) measuring the amount of amplification product or a quantityindicating the amount of amplification product for said target nucleicacid and said control nucleic acid during said amplification in relationto an increasing progress parameter (cycle_(k)) representing theprogress of said amplification process; d) determining a characteristicvalue (nT_(i)) of said progress parameter for said target nucleic acidon basis of measurement results related to the amount of amplificationproduct for said target nucleic acid; e) if certain pre-definedconditions referring to the measured amount of amplification product forsaid control nucleic acid apply, determining a characteristic value(nQ_(i)) of said progress parameter for said control nucleic acid onbasis of measurement results related to the amount of amplificationproduct for said control nucleic acid, wherein said pre-definedconditions are selected from at least one of the following: i) thecharacteristic value for said control nucleic acid falls short of apredefined threshold value, ii) the characteristic value for said targetnucleic acid exceeds a predefined threshold value, iii) thecharacteristic value for said control nucleic acid falls short of athreshold value defined in relation and greater than the characteristicvalue for said target nucleic acid, iv) the amount of amplificationproduct or the quantity indicating the amount of amplification productfor said control nucleic acid as measured or estimated for a final stageor near final stage of the amplification process exceeds of a predefinedminimum plateau value, v) the amount of amplification product or thequantity indicating the amount of amplification product for said controlnucleic acid as measured or estimated for a momentary state of theamplification process associated to the characteristic value for thetarget nucleic acid exceeds a predefined threshold value, the amount ofamplification product, or the quantity indicating the amount ofamplification product for said target nucleic acid as measured orestimated for said momentary state of the amplification process; f)selecting between a plurality of quantification schemes according to atleast one predefined selection criterion, said selection being effectedon basis of at least one of said measurement results related to theamount of amplification product for said target nucleic acid,measurement results related to the amount of amplification product forsaid control nucleic acid, and said characteristic value or values,wherein at least one quantification scheme of a first type provides fora quantification of the original amount (T_(0i)) of target nucleic acidin said test sample on basis of the characteristic value (nT_(i)) forsaid target nucleic acid, the characteristic value for said controlnucleic acid (nQ_(i)) and associated reference data, wherein at leastone quantification scheme of a second type provides for a quantificationof the original amount (T_(0i)) of target nucleic acid in said testsample on basis of the characteristic value (nT_(i)) for said targetnucleic acid and associated reference data without reference to anycharacteristic value (nQ_(i)) for said control nucleic acid; and g)quantifying the original amount of target nucleic acid (T_(0i)) in saidtest sample according to the selected quantification scheme on basis ofat least the characteristic value (nT_(i)) determined for said targetnucleic acid.
 2. The method of claim 1, wherein selecting between aplurality of quantification schemes according to at least one predefinedselection criterion includes selecting a quantification scheme of thesecond type if at least one of the following conditions applies: i) nocharacteristic value (nQ_(i)) for said control nucleic acid wasdetermined, ii) the characteristic value for said control nucleic acidexceeds a predefined threshold value, iii) the characteristic value forsaid target nucleic acid (nT_(i)) falls short of a predefined thresholdvalue, iv) the characteristic value for said control nucleic acidexceeds the characteristic value for said target nucleic acid by atleast a predefined amount, v) the amount of amplification product or thequantity (afi(n)) indicating the amount of amplification product forsaid control nucleic acid as measured or estimated for a final stage ornear final stage of the amplification process falls short of apredefined minimum plateau value (afin_(ref)), vi) the amount ofamplification product or the quantity indicating the amount ofamplification product for said control nucleic acid as measured orestimated for a momentary state of the amplification process associatedto the characteristic value for the target nucleic acid falls short of apredefined threshold value or of the amount of amplification product orthe quantity indicating the amount of amplification product for saidtarget nucleic acid as measured or estimated for said momentary state ofthe amplification process.
 3. The method according to claim 1, whereinsaid reference data associated to said quantification scheme of thefirst type are calibration data determined by A) providing at least onestandard together with at least one internal control in a common sample,said standard comprising a defined initial amount (T_(0i)) of a standardnucleic acid, said internal control comprising a defined initial amount(Q₀) of a control nucleic acid, said standard nucleic acid and saidcontrol nucleic acid being different; B) amplifying said standard andsaid internal control within said sample in a common nucleic acidamplification process; C) directly or indirectly measuring the amount ofamplification product or a quantity indicating the amount ofamplification product for said standard nucleic acid and said controlnucleic acid during said amplification in relation to an increasingprogress parameter (cycle_(k)) representing the progress of saidamplification process; D) determining a characteristic value (nT_(i)) ofsaid progress parameter for said standard nucleic acid on basis ofmeasurement results related to the amount of amplification product forsaid standard nucleic acid; E) determining a characteristic value(nQ_(i)) of said progress parameter for said control nucleic acid onbasis of measurement results related to the amount of amplificationproduct for said control nucleic acid; and H) relating said initialamount of standard nucleic acid (T_(0i)) on the one hand and saidcharacteristic values (nT_(i), nQ_(i)) on the other hand with referenceto said quantification scheme of the first type to provide saidcalibration data (a, b, c) associated to said quantification scheme ofthe first type.
 4. The method according to claim 1, wherein saidreference data associated to said quantification scheme of the secondtype are calibration data determined by: AA) providing at least onestandard in a sample, said standard comprising a defined initial amountof a standard nucleic acid; BB) amplifying said standard in a nucleicacid amplification process; CC) directly or indirectly measuring theamount of amplification product or a quantity indicating the amount ofamplification product for said standard nucleic acid during saidamplification in relation to an increasing progress parameterrepresenting the progress of said amplification process; DD) determininga characteristic value of said progress parameter for said standardnucleic acid on basis of measurement results related to the amount ofamplification product for said standard nucleic acid; and HH) relatingsaid initial amount (T_(0i)) of standard nucleic acid on the one handand said characteristic value (nT_(i)) on the other hand with referenceto said quantification scheme of the second type to provide saidcalibration data (A, B, C; T_(ref), nT_(ref)) associated to saidquantification scheme of the second type.
 5. The method according toclaim 4, wherein said reference data associated to said quantificationscheme of the second type are calibration data determined or provided onbasis of steps A) to D) and on basis of HH) relating said initial amount(T_(0i)) of standard nucleic acid on the one hand and saidcharacteristic value (nT_(i)) associated to said standard nucleic acidon the other hand with reference to said quantification scheme of thesecond type to provide said calibration data (A, B, C; T_(ref),nT_(ref)) associated to said quantification scheme of the second type.6. The method according to claim 3, wherein said standard is an externalstandard, said sample being different from said test sample.
 7. Themethod according to claim 3, wherein said defined initial amount (Q_(i))of said control nucleic acid being amplified together with said standardnucleic acid is the same as said defined initial amount (Q₀) of saidcontrol nucleic acid being amplified together with said target nucleicacid.
 8. The method according to claim 3, wherein said standard nucleicacid corresponds to said target nucleic acid.
 9. The method according toclaim 3, wherein said calibration data (a, b, c) associated to saidquantification scheme of the first type or said calibration data (A, B,C; T_(ref), nT_(ref)) associated to said quantification scheme of thesecond type are provided together with constituents of a quantificationkit.
 10. The method according to claim 3, wherein in step A) a dilutionseries (T_(0i) (i=1, . . . , S)) of said standard nucleic acid isprovided, each dilution within a respective sample together with saidinternal control, wherein steps B) to E) are effected with respect toall samples of said dilution series, and wherein step H) comprises:relating the initial amounts (T_(0i)) of standard nucleic acid of saidsamples and the characteristic values (nT_(i), nQ_(i)) determined forsaid samples with reference to said quantification scheme of the firsttype to provide said calibration data (a, b, c) associated to saidquantification scheme of the first type.
 11. The method according toclaim 10, wherein said reference data associated to said quantificationscheme of the second type are calibration data determined on basis ofsteps A) to D) and on basis of HH) relating the initial amount (T_(0j))of standard nucleic acid of a selected or predefined one of said samplesand the characteristic value (nT_(j)) associated to said standardnucleic acid determined for said sample being selected with reference tosaid quantification scheme of the second type to provide saidcalibration data (T_(ref), nT_(ref)) associated to said quantificationscheme of the second type.
 12. The method according to claim 11, whereinsaid reference data associated to said quantification scheme of thesecond type are calibration data determined or provided on basis ofsteps A) to D) and on basis of HH) relating the initial amounts (T_(0i))of standard nucleic acid of said samples and the characteristic values(nT_(i)) associated to said standard nucleic acid determined for saidsamples with reference to said quantification scheme of the second typeto provide said calibration data (A, B, C) associated to saidquantification scheme of the second type.
 13. The method according toclaim 4, wherein in step AA) only one sample including a selecteddefined initial amount (T_(0j)) of said standard nucleic acid isprovided.
 14. The method according to claim 4, wherein in step AA) adilution series (T_(0i) (i=1, . . . , S)) of said standard nucleic acidis provided, each dilution being within a respective sample, whereinsteps BB) to DD) are effected with respect to all samples of saiddilution series, and wherein step HH) comprises: relating the initialamounts (T_(0i)) of standard nucleic of said samples and thecharacteristic values (nT_(i)) determined for said samples withreference to said quantification scheme of the second type to providesaid calibration data (A, B, C) associated to said quantification schemeof the second type.
 15. The method according to claim 9, wherein thecalibration data associated to said quantification scheme of the secondtype include a fixed amplification efficiency (ε) or wherein in step g)the calibration data associated to said quantification scheme of thesecond type are used together with a fixed amplification efficiency (ε)for the quantification of the original amount of target nucleic in saidtest sample according to the quantification scheme of the second type.16. The method according to claim 15, wherein a theoreticalamplification efficiency (ε) of said amplification process is used assaid fixed amplification efficiency.
 17. The method according to claim1, wherein said amplification process is effected in cycles and a cyclenumber indicating the number of elapsed cycles is used as progressparameter.
 18. The method according to claim 1, wherein at least one ofstep d) and step e) comprises: deriving a growth curve from therespective measurement results, identifying a characteristic of therespective growth curve or of a derivative calculated of the respectivegrowth curve, and determining the characteristic value (nT_(i); nQ_(i))associated with said characteristic.
 19. The method according to claim18, wherein the characteristic of the respective growth curvecorresponds to a crossing of a threshold by the growth curve, saidthreshold being predefined to represent an unnormalized growth value orbeing determined on basis of respective measurement results to representa normalized growth value.
 20. The method according to claim 1, whereinaccording to said quantification scheme of the first type a secondarycharacteristic value (Δn_(i)) is determined from said characteristicvalue (nT_(i)) for said target nucleic acid or standard nucleic acid,respectively, and said characteristic value (nQ_(i)) of said controlnucleic acid, said secondary characteristic value representing a director indirect measure of at least one of the amplification and theoriginal amount (T_(0i)) of said target nucleic acid or initial amount(T_(0i)) of said standard nucleic acid, respectively, relative to atleast one of the amplification and the defined initial amount (Q₀) ofsaid control nucleic acid, and wherein the original amount (T_(0i)) oftarget nucleic acid is quantified on basis of said secondarycharacteristic value (Δn_(i)) and said reference data (a, b, c)associated thereto.
 21. The method according to claim 20, wherein stepH) comprises: relating said initial amount (T_(0i)) of standard nucleicacid and said secondary characteristic value (Δn_(i)) with reference tosaid quantification scheme of the first type to provide said calibrationdata (a, b, c) associated to said quantification scheme of the firsttype.
 22. The method according to claim 1, wherein according to saidquantification scheme of the first type a difference value (Δn_(i))representing the difference between the characteristic value (nT_(i))for said target nucleic acid or standard nucleic acid, respectively, andthe characteristic value (nQ_(i)) of said control nucleic acid isdetermined, and wherein the original amount (T_(0i)) of target nucleicacid is quantified on basis of said difference value (Δn_(i)) and saidreference data (a, b, c) associated thereto.
 23. The method according toclaim 22, wherein step H) comprises: relating said initial amount ofstandard nucleic acid and said difference value with reference to saidquantification scheme of the first type to provide said calibration dataassociated to said quantification scheme of the first type.
 24. Themethod according to claim 1, wherein said amplification processcomprises a polymerase chain reaction (PCR) process or wherein saidamplification process comprises or is part of a reverse transcriptasepolymerase chain reaction (RT-PCR) process.
 25. The method according toclaim 24, wherein in said amplification process said target nucleic acidor standard nucleic acid and said control nucleic acid are competitivelyamplified on basis of the same primers for the target nucleic acid orstandard nucleic acid and for said control nucleic acid.
 26. The methodaccording to claim 1, wherein selecting between a plurality ofquantification schemes includes selecting between a quantification ofsaid target nucleic acid in said test sample and a determination ofpresence or non-presence of said target nucleic acid in said testsample, and if selected, determining the presence or non-presence ofsaid target nucleic acid in said test sample on the basis of measurementresults obtained in step c).
 27. Apparatus for quantification of atleast one target nucleic acid in a test sample or in a plurality of testsamples, the apparatus comprising: an amplification unit for effecting anucleic acid amplification process with respect to at least one testsample; a detection unit for measuring, at a plurality of differenttimes during said nucleic acid amplification process effected by saidamplification unit, at least two signals being related to a respectivenucleic acid which is amplified in the amplification process, thedetection mechanism being adapted to independently measure at least onefirst signal related only to a first nucleic acid and at least onesecond signal related only to a second nucleic acid or to measure atleast one first signal and at least one second signal from which firstdata related only to a first nucleic acid and second data related onlyto a second nucleic can be calculated; a controller in communicationwith said amplification unit and said detection mechanism; wherein saidcontroller is configured to perform operations comprising: bb)controlling the amplification unit to effect an amplification withrespect to at least one respective test sample; cc) controlling thedetection unit to directly or indirectly measure the amount ofamplification product or a quantity indicating the amount ofamplification product for at least two different nucleic acids, namelywith respect to at least one first nucleic acid and with respect to atleast one second nucleic acid; dd) determining a characteristic value(nT_(i)) of said progress parameter for said first nucleic acid on basisof measurement results related to the amount of amplification productfor said first nucleic acid; ee) if certain pre-defined conditionsreferring to the measured amount of amplification product for saidsecond nucleic acid apply, determining a characteristic value (nQ_(i))of said progress parameter also for said second nucleic acid on basis ofmeasurement results related to the amount of amplification product forsaid second nucleic acid, wherein said pre-defined conditions areselected from at least one of the following: i) the characteristic valuefor said second nucleic acid falls short of a predefined thresholdvalue, ii) the characteristic value for said first nucleic acid exceedsa predefined threshold value, iii) the characteristic value for saidsecond nucleic acid falls short of a threshold value defined in relationand greater than the characteristic value for said first nucleic acid,iv) the amount of amplification product or the quantity indicating theamount of amplification product for said second nucleic acid as measuredor estimated for a final stage or near final stage of the amplificationprocess exceeds of a predefined minimum plateau value, v) the amount ofamplification product or the quantity indicating the amount ofamplification product for said second nucleic acid as measured orestimated for a momentary state of the amplification process associatedto the characteristic value for the first nucleic acid exceeds apredefined threshold value, the amount of amplification product, or thequantity indicating the amount of amplification product for said firstnucleic acid as measured or estimated for said momentary state of theamplification process; ff) selecting between a plurality ofquantification schemes according to at least one predefined selectioncriterion, said selection being effected directly or indirectly on basisof at least one of said measurement results related to the amount ofamplification product for said first nucleic acid, measurement resultsrelated to the amount of amplification product for said second nucleicacid and said characteristic value or values, wherein at least onequantification scheme of a first type provides for a quantification ofthe original amount (T_(0i)) of first nucleic acid in said test sampleon basis of the characteristic value (nT_(i)) for said first nucleicacid, the characteristic value (nQ_(i)) for said second nucleic acid andassociated reference data, wherein at least one quantification scheme ofa second type provides for a quantification of the original amount(T_(0i)) of first nucleic acid in said test sample on basis of thecharacteristic value (nT_(i)) for said first nucleic acid and associatedreference data without reference to any characteristic value (nQ_(i))for said second nucleic acid, gg) quantification of the original amount(T_(0i)) of first nucleic acid in said test sample according to theselected quantification scheme on basis of at least the characteristicvalue (nT_(i)) determined for said first nucleic acid, jj) providingquantification data which include said original amount (T_(0i)) of firstnucleic acid to represent the original amount (T_(0i)) of target nucleicin said test sample.
 28. A computer program product, embodied intangible medium, the product comprising instructions executable by anapparatus for quantification of at least one target nucleic acid in atest sample or in a plurality of test samples, the apparatus comprising:an amplification unit for effecting a nucleic acid amplification processwith respect to at least one test sample; a detection mechanism formeasuring, at a plurality of different times during said nucleic acidamplification process effected by said amplification unit, at least twosignals being related to a respective nucleic acid which is amplified inthe amplification process, the detection mechanism being adapted toindependently measure at least one first signal related only to a firstnucleic acid and at least one second signal related only to a secondnucleic acid or to measure at least one first signal and at least onesecond signal from which first data related only to a first nucleic acidand second data related only to a second nucleic can be calculated; acontroller in communication with said amplification unit and saiddetection mechanism; wherein said controller in response to saidinstructions performs operations comprising: bb) controlling theamplification unit to effect an amplification with respect to at leastone respective test sample; cc) controlling the detection unit todirectly or indirectly measure the amount of amplification product or aquantity indicating the amount of amplification product for at least twodifferent nucleic acids, namely with respect to at least one firstnucleic acid and with respect to at least one second nucleic acid; dd)determining a characteristic value (nT_(i)) of said progress parameterfor said first nucleic acid on basis of measurement results related tothe amount of amplification product for said first nucleic acid; ee) ifcertain pre-defined conditions referring to the measured amount ofamplification product for said second nucleic acid apply, determining acharacteristic value (nQ_(i)) of said progress parameter also for saidsecond nucleic acid on basis of measurement results related to theamount of amplification product for said second nucleic acid, whereinsaid pre-defined conditions are selected from at least one of thefollowing: i) the characteristic value for said second nucleic acidfalls short of a predefined threshold value, ii) the characteristicvalue for said first nucleic acid exceeds a predefined threshold value,iii) the characteristic value for said second nucleic acid falls shortof a threshold value defined in relation and greater than thecharacteristic value for said first nucleic acid, iv) the amount ofamplification product or the quantity indicating the amount ofamplification product for said second nucleic acid as measured orestimated for a final stage or near final stage of the amplificationprocess exceeds of a predefined minimum plateau value, v) the amount ofamplification product or the quantity indicating the amount ofamplification product for said second nucleic acid as measured orestimated for a momentary state of the amplification process associatedto the characteristic value for the first nucleic acid exceeds apredefined threshold value, the amount of amplification product, or thequantity indicating the amount of amplification product for said firstnucleic acid as measured or estimated for said momentary state of theamplification process; ff) selecting between a plurality ofquantification schemes according to at least one predefined selectioncriterion, said selection being effected directly or indirectly on basisof at least one of said measurement results related to the amount ofamplification product for said first nucleic acid, measurement resultsrelated to the amount of amplification product for said second nucleicacid and said characteristic value or values, wherein at least onequantification scheme of a first type provides for a quantification ofthe original amount (T_(0i)) of first nucleic acid in said test sampleon basis of the characteristic value (nT_(i)) for said first nucleicacid, the characteristic value (nQ_(i)) for said second nucleic acid andassociated reference data, wherein at least one quantification scheme ofa second type provides for a quantification of the original amount(T_(0i)) of first nucleic acid in said test sample on basis of thecharacteristic value (nT_(i)) for said first nucleic acid and associatedreference data without reference to any characteristic value (nQ_(i))for said second nucleic acid, gg) quantification of the original amount(T_(0i)) of first nucleic acid in said test sample according to theselected quantification scheme on basis of at least the characteristicvalue (nT_(i)) determined for said first nucleic acid, jj) providingquantification data which include said original amount (T_(0i)) of firstnucleic acid to represent the original amount (T_(0i)) of target nucleicin said test sample.
 29. A server computer system storing the computerprogram product according to claim 28 for downloading via acommunication link.